Feed additive composition

ABSTRACT

A feed additive composition comprising a direct fed microbial in combination with a protease, a xylanase, an amylase and a phytase, and a method for improving the performance of a subject or for improving digestibility of a raw material in a feed (e.g. nutrient digestibility, such as amino acid digestibility), or for improving nitrogen retention, or for avoiding the negative effects of necrotic enteritis or for improving feed conversion ratio (FCR) or for improving weight gain in a subject or for improving feed efficiency in a subject or for modulating (e.g. improving) the immune response of the subject or for promoting the growth of beneficial bacteria in the gastrointestinal tract of a subject, which method comprising administering to a subject a direct fed microbial in combination with a protease, a xylanase, an amylase and a phytase.

FIELD OF INVENTION

The present invention relates to methods for improving feed compositionsusing a direct fed microbial in combination with a specific combinationof enzymes, and to a feed additive composition comprising a direct fedmicrobial in combination with a specific combination of enzymes. Thepresent invention further relates to uses and kits.

BACKGROUND OF THE INVENTION

Supplemental enzymes are used as additives to animal feed, particularlypoultry and swine feeds, as a means to improve nutrient utilization andproduction performance characteristics. Enzyme blends are available toimprove the nutritional value of diets containing soybean meal, animalprotein meals, or high fibre food by-products.

The concept of direct fed microbials (DFM) involves the feeding ofbeneficial microbes to animals, such as dairy cattle when they are underperiods of stress (disease, ration changes, environmental or productionchallenges). Probiotics is another term for this category of feedadditives. Probiotics or DFM have been shown to improve animalperformance in controlled studies. DFM including direct fed bacteria andor yeast-based products.

Although combinations of DFMs with some enzymes have been contemplated,the interaction between DFMs and enzyme has never been fully understood.The present invention relates to novel specific combinations whichsurprisingly significantly improve production performancecharacteristics of animals.

SUMMARY OF INVENTION

A seminal finding of the present invention is that a DFM in combinationwith a protease, xylanase, amylase and phytase has significantbeneficial effects on the performance of an animal.

In particular, a seminal finding of the present invention is that a DFMin combination with a protease, xylanase, amylase and phytase hassignificant beneficial effects on the performance of an animal,including improving one or more of the following: feed conversion ratio(FCR), ability to digest a raw material (e.g. nutrient digestibility,such as amino acid digestibility), nitrogen retention, survival, carcassyield, growth rate, weight gain, feed efficiency animals resistance tonecrotic enteritis, immune response of the subject, or the growth ofbeneficial bacteria in the gastrointestinal tract of a subject.

Another surprising effect of the present invention is that it can reducenutrient excretion in manure (e.g. reduce nitrogen and phosphorus)content of a subject's manure.

In one aspect, the present invention provides a feed additivecomposition comprising (or consisting essentially of or consisting of) adirect fed microbial in combination with a protease, a xylanase, anamylase and a phytase.

In another aspect, the present invention provides a method for improvingthe performance of a subject or for improving digestibility of a rawmaterial in a feed (e.g. nutrient digestibility, such as amino aciddigestibility), or for improving nitrogen retention, or for avoiding thenegative effects of necrotic enteritis or for improving feed conversionratio (FCR) or for improving weight gain in a subject or for improvingfeed efficiency in a subject or for modulating (e.g. improving) theimmune response of the subject or for promoting the growth of beneficialbacteria in the gastrointestinal tract of a subject or for reducingpopulations of pathogenic bacteria in the gastrointestinal tract of asubject, or for reducing nutrient excretion in manure, which methodcomprising administering to a subject a direct fed microbial incombination with a protease, a xylanase, an amylase and a phytase.

A yet further aspect of the present invention is use of a direct fedmicrobial in combination with a protease, a xylanase, an amylase and aphytase for improving the performance of a subject or for improvingdigestibility of a raw material in a feed (e.g. nutrient digestibility,such as amino acid digestibility) or for improving nitrogen retention)or for avoiding the negative effects of necrotic enteritis or forimproving feed conversion ratio (FCR) or for improving weight gain in asubject or for improving feed efficiency in a subject or for modulating(e.g. improving) the immune response of the subject or for promoting thegrowth of beneficial bacteria in the gastrointestinal tract of a subjector for reducing populations of pathogenic bacteria in thegastrointestinal tract of a subject, or for reducing nutrient excretionin manure.

In a further aspect of the present invention there is provided a kitcomprising a direct fed microbial, a protease, a xylanase, an amylase, aphytase (and optionally at least one vitamin and/or optionally at leastone mineral) and instructions for administration.

In another aspect the present invention provides a method of preparing afeed additive composition, comprising admixing a direct fed microbialwith a protease, a xylanase, an amylase and a phytase and (optionally)packaging.

In a yet further aspect the present invention provides feed or feedstuffcomprising a feed additive composition comprising (or consistingessentially of or consisting of) a direct fed microbial in combinationwith a protease, a xylanase, an amylase and a phytase.

A premix comprising a feed additive composition comprising (orconsisting essentially of or consisting of) a direct fed microbial incombination with a protease, a xylanase, an amylase and a phytase, andat least one mineral and/or at least one vitamin.

In another aspect, the present invention provides a method of preparinga feedstuff comprising admixing a feed component with a feed additivecomposition comprising (or consisting essentially of or consisting of) adirect fed microbial in combination with a protease, a xylanase, anamylase and a phytase.

In a further aspect, the present invention relates to a feed additivecomposition for preventing and/or treating coccidiosis and/or necroticenteritis in a subject.

The present invention yet further provides a method of preventing and/ortreating necrotic enteritis and/or coccidiosis wherein an effectiveamount of a feed additive composition according to the present inventionis administered to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that a combination of DFM (Enviva Pro® available fromDanisco A/S) with a combination of a xylanase (e.g. an endo-xylanasefrom Trichoderma xylanase), an amylase (e.g. a Bacillus licheniformisalpha-amylase), a protease (e.g. Bacillus subtilis protease) and aphytase (e.g. 500 FTU/kg of Phyzyme XP (an E. coli phytase) availablefrom Danisco A/S) significantly improved (reduced) necrotic enteritislesion scores in the gut of the animals compared with the challengedcontrol. In some embodiments the xylanase, amylase and protease mayformulated together in AxtraXAP® [containing 2000 XU/kg feed ofxylanase; 200 AU/kg feed of amylase and 4000 PU/kg feed of protease]also available from Danisco A/S).

FIG. 2 shows that a combination of (Enviva Pro® available from DaniscoA/S) with a combination of a xylanase (e.g. an endo-xylanase fromTrichoderma xylanase), an amylase (e.g. a Bacillus licheniformisalpha-amylase), a protease (e.g. Bacillus subtilis protease) and aphytase (e.g. 500 FTU/kg of Phyzyme XP (an E. coli phytase) availablefrom Danisco A/S) significantly improved Body weight gain (BW gain) inbroiler chickens challenged with Clostridium perfringens compared withthe challenged control—even resulting in a BW gain which was improvedover a negative control (i.e. an unchallenged control). This wassignificantly better than any other combinations of enzymes such aseither amylase and phytase or protease and phytase, and significantlybetter than DFM applied on the challenged control. In some embodimentsthe xylanase, amylase and protease may formulated together in AxtraXAP®[containing 2000 XU/kg feed of xylanase; 200 AU/kg feed of amylase and4000 PU/kg feed of protease] also available from Danisco A/S). PooledSEM=28.6

FIG. 3 shows a combination of (Enviva Pro® available from Danisco A/S)with a combination of a xylanase (e.g. an endo-xylanase from Trichodermaxylanase), an amylase (e.g. a Bacillus licheniformis alpha-amylase), aprotease (e.g. Bacillus subtilis protease) and a phytase (e.g. 500FTU/kg of Phyzyme XP (an E. coli phytase) available from Danisco A/S)significantly improved feed conversion ratio (FCR) (g feed intake/g BWgain) in broiler chickens challenged with Clostridium perfringens to thelevel of unchallenged birds. This was significantly better than othercombinations of enzymes with the DFM such as either amylase and phytaseor protease and phytase. In some embodiments the xylanase, amylase andprotease may formulated together in AxtraXAP® [containing 2000 XU/kgfeed of xylanase; 200 AU/kg feed of amylase and 4000 PU/kg feed ofprotease] also available from Danisco A/S).

FIG. 4 shows relative mRNA expression of IFN-g used as marker ofinflammation in the intestine, and shows that a combination of DFM(Enviva Pro®) with a combination of xylanase, amylase, protease andphytase (Avizyme 1502® available from Danisco A/S+500 FTU/kg of PhyzymeXP (an E. coli phytase) increased IFN-g expression at 11 days andreduced it at 20 days.

FIG. 5 shows apparent ileal digestible energy (mCal/kg) and shows that acombination of DFM (Enviva Pro®) with a xylanase, amylase, protease andphytase (two different enzyme mixes were used the first was Avizyme1502® available from Danisco A/S+500 FTU/kg of Phyzyme XP (an E. coliphytase); and the second was AxtraXAP [containing 2000 XU/kg feed ofxylanase; 200 AU/kg feed of amylase and 4000 PU/kg feed of protease]also available from Danisco A/S+500 FTU/kg of Phyzyme XP (an E. coliphytase) significantly improved energy digestibility effects.

FIG. 6 shows amino acid digestibility significantly improved with acombination of DFM (Enviva Pro®) with a xylanase, amylase, protease andphytase. The improvement of digestibility of the undigested fractions ofamino acid at the ileal level with a combination of DMF with xylanase,amylase, protease and phytase was greater than the improvement of DFMalone or the combination of xylanase, amylase, protease and phytasewithout DFM.

FIG. 7 shows energy digestibility improved with a combination of DFM(Enviva Pro®) with a xylanase, amylase, protease and phytase.

FIG. 8 shows nitrogen-corrected apparent metabolizable energy AMEn ofdietary treatments fed to 17 to 21-d-old broiler chickens.

FIG. 9 shows that a combination of DFM (Enviva Pro®) with a xylanase,amylase, protease and phytase (two different enzyme mixes were used thefirst was Avizyme 1502® available from Danisco A/S+500 FTU/kg of PhyzymeXP (an E. coli phytase); and the second was AxtraXAP also available fromDanisco A/S+500 FTU/kg of Phyzyme XP (an E. coli phytase) significantlyimproved nitrogen retention.

FIG. 10 shows that a combination of DFM (Enviva Pro®) with a xylanase,amylase, protease and phytase (Avizyme 1502® available from DaniscoA/S+Phyzyme XP (an E. coli phytase)) significantly reduces the mRNAabundance of MUC-2 in the ileal mucosal scrapings at day 14 treated withan overdosed coccidian vaccine at hatch, compared to the challenged andunchallenged control treatments.

FIG. 11 shows the amino acid sequence (SEQ ID No. 1) of a pepsinresistant alpha amylase from Bacillus licheniformis.

FIG. 12 shows the nucleotide sequence (SEQ ID No. 2) of a pepsinresistant alpha amylase from Bacillus licheniformis.

FIG. 13 shows the amino acid sequence (SEQ ID No. 3) of a pepsinresistant alpha amylase from Trichoderma reesei.

FIG. 14 shows the nucleotide sequence (SEQ ID No. 4) of a pepsinresistant alpha amylase from Trichoderma reesei.

FIG. 15 shows feed conversion ratio of broiler chickens at 48 d of age.

FIG. 16 shows a heat map of expression profiles of genes of interest forall treatments for jejunum at 23 days of age.

Unchallenged control=Unchallenged Control+phytase

CC=Challenged Control+phytase

CC+Amylase=Challenged Control+phytase+amylase

CC+XAP=Challenged Control+phytase+xylanase+amylase+protease

CC+EP=Challenged Control+phytase+Enviva Pro

CC+EP+Amylase=Challenged Control+phytase+amylase+Enviva Pro

CC+EP+XAP=Challenged Control+phytase+xylanase+amylase+protease+EnvivaPro.

FIG. 17 shows a heat map of expression profile of chicken alpha amylasefor all treatments in pancreas at 23 days of age.

Unchallenged control=Unchallenged Control+phytase

CC=Challenged Control+phytase

CC+Amylase=Challenged Control+phytase+amylase

CC+XAP=Challenged Control+phytase+xylanase+amylase+protease

CC+EP=Challenged Control+phytase+Enviva Pro

CC+EP+Amylase=Challenged Control+phytase+amylase+Enviva Pro

CC+EP+XAP=Challenged Control+phytase+xylanase+amylase+protease+EnvivaPro.

FIG. 18 shows apparent metabolizable energy corrected by nitrogenretention (AME_(n)) of 21 d old broiler chickens. Effect of DFM;P<0.001; Effect of Enzyme; P<0.001; Effect of DFM×Enzyme; P=0.27; PooledSEM=32 kcal.

FIG. 19 shows feed conversion ratio (FCR) of broiler chickens in anecrotic enteritis challenge model (Pooled SEM: 0.015).

FIG. 20 shows relative proportion of Lactobacillus spp. at 21 d injejunum in broiler chickens, ChSq<0.0001.

DETAILED DESCRIPTION OF THE INVENTION

Preferably each of the enzymes used in the present invention areexogenous to the DFM. In other words the enzymes are preferably added toor admixed with the DFM.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, NewYork (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materialsdisclosed herein, and any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of this disclosure. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, any nucleic acidsequences are written left to right in 5′ to 3′ orientation; amino acidsequences are written left to right in amino to carboxy orientation,respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of this disclosure which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

Amino acids are referred to herein using the name of the amino acid, thethree letter abbreviation or the single letter abbreviation.

The term “protein”, as used herein, includes proteins, polypeptides, andpeptides.

As used herein, the term “amino acid sequence” is synonymous with theterm “polypeptide” and/or the term “protein”. In some instances, theterm “amino acid sequence” is synonymous with the term “peptide”. Insome instances, the term “amino acid sequence” is synonymous with theterm “enzyme”.

The terms “protein” and “polypeptide” are used interchangeably herein.In the present disclosure and claims, the conventional one-letter andthree-letter codes for amino acid residues may be used. The 3-lettercode for amino acids as defined in conformity with the IUPACIUB JointCommission on Biochemical Nomenclature (JCBN). It is also understoodthat a polypeptide may be coded for by more than one nucleotide sequencedue to the degeneracy of the genetic code.

Other definitions of terms may appear throughout the specification.Before the exemplary embodiments are described in more detail, it is tounderstand that this disclosure is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present disclosure will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin this disclosure. The upper and lower limits of these smallerranges may independently be included or excluded in the range, and eachrange where either, neither or both limits are included in the smallerranges is also encompassed within this disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anenzyme” includes a plurality of such candidate agents and reference to“the feed” includes reference to one or more feeds and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that such publicationsconstitute prior art to the claims appended hereto.

The enzymes for use in the present invention can be produced either bysolid or submerged culture, including batch, fed-batch andcontinuous-flow processes. Culturing is accomplished in a growth mediumcomprising an aqueous mineral salts medium, organic growth factors, thecarbon and energy source material, molecular oxygen, and, of course, astarting inoculum of one or more particular microorganism species to beemployed.

Direct Fed Microbial (DFM)

The term “microbial” herein is used interchangeably with“microorganism”.

Preferably the DFM comprises a viable microorganism. Preferably the DFMcomprises a viable bacterium or a viable yeast or a viable fungi.

In one preferred embodiment the DFM comprises a viable bacteria.

The term “viable microorganism” means a microorganism which ismetabolically active or able to differentiate.

In one embodiment the DFM may be a spore forming bacterium and hence theterm DFM may be comprised of or contain spores, e.g. bacterial spores.Therefore in one embodiment the term “viable microorganism” as usedherein may include microbial spores, such as endospores or conidia.

In another embodiment the DFM in the feed additive composition accordingto the present invention is not comprised of or does not containmicrobial spores, e.g. endospores or conidia.

The microorganism may be a naturally occurring microorganism or it maybe a transformed microorganism. The microorganism may also be acombination of suitable microorganisms.

In some aspects, the DFM according to the present invention may be oneor more of the following: a bacterium, a yeast or a fungi.

Preferably the DFM according to the present invention is a probioticmicroorganism.

In the present invention, the term direct fed microbial (DFM)encompasses direct fed bacteria, direct fed yeast, direct fed yeast andcombinations thereof.

Preferably the DFM is a direct fed bacterium.

Preferably the DFM is a combination comprising two or more bacteria,e.g. three or more or four or more; or the DFM is a combinationcomprising two or more bacterial strains, e.g. three or more or four ormore.

Preferably the bacterium or bacteria is or are isolated.

Suitably the DFM may comprise a bacterium from one or more of thefollowing genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus,Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,Propionibacterium, Bifidobacterium, Clostridium and Megasphaera andcombinations thereof.

In one embodiment the DFM may be selected from the following Bacillusspp: Bacillus subtilis, Bacillus cereus, Bacillus licheniformis andBacillus amyloliquefaciens.

In one embodiment the DFM may be a combination comprising two or moreBacillus strains.

In one embodiment the DFM may be a combination of two or more theBacillus subtilis strains 3A-P4 (PTA-6506); 15A-P4 (PTA-6507); 22C-P1(PTA-6508); 2084 (NRRL B-500130); LSSA01 (NRRL-B-50104); BS27 (NRRLB-50105); BS 18 (NRRL B-50633); and BS 278 (NRRL B-50634).

Strains 3A-P4 (PTA-6506), 15A-P4 (PTA-6507) and 22C-P1 (PTA-6508) arepublically available from American Type Culture Collection (ATCC).

Strains 2084 (NRRL B-500130); LSSA01 (NRRL-B-50104); BS27 (NRRL B-50105)are publically available from the Agricultural Research Service CultureCollection (NRRL). Strain Bacillus subtilis LSSA01 is sometimes referredto as B. subtilis 8.

These strains are taught in U.S. Pat. No. 7,754,469 B2.

Bacillus subtilis BS 18 and Bacillus subtilis BS 278 were deposited byAndy Madisen of W227 N752 Westmound Dr. Waukesha, Wis. 53186, USA orDanisco USA Inc. of W227 N752 Westmound Dr. Waukesha, Wis. 53186, USAunder the Budapest Treaty at the Agricultural Research Service CultureCollection (NRRL) at 1815 North University Street, Peoria, Ill. 61604,United States of America, under deposit numbers NRRL B-50633 and NRRLB-50634, respectively on 9 Jan. 2012.

Andy Madisen of W227 N752 Westmound Dr. Waukesha, Wis. 53186, USA andDanisco USA Inc. of W227 N752 Westmound Dr. Waukesha, Wis. 53186, USAauthorise Danisco A/S of Langebrogade 1, PO Box 17, DK-1001, CopenhagenK, Denmark to refer to these deposited biological materials in thispatent application and have given unreserved and irrevocable consent tothe deposited material being made available to the public.

In some embodiments the DFM may be a combination comprising the Bacillussubtilis strains as detailed in the table below:

B. subtilis strain Bs Bs Bs Bs Bs Bs Bs 2084 8 (LSSAO1) 3A-P4 15A-P4 27818 22C-P1 DFM X X X X Combi- X X X nation X X X comprises X X X X X X XX X X X X X X X X X X

In one embodiment the DFM may be selected from the following Lactococcusspp: Lactococcus cremoris and Lactococcus lactis and combinationsthereof.

In one embodiment the DFM may be selected from the followingLactobacillus spp: Lactobacillus buchneri, Lactobacillus acidophilus,Lactobacillus casei, Lactobacillus kefiri, Lactobacillus bifidus,Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei,Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacilluscurvatus, Lactobacillus bulgaricus, Lactobacillus sakei, Lactobacillusreuteri, Lactobacillus fermentum, Lactobacillus farciminis,Lactobacillus lactis, Lactobacillus delbreuckii, Lactobacillusplantarum, Lactobacillus paraplantarum, Lactobacillus farciminis,Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri,Lactobacillus johnsonii and Lactobacillus jensenii, and combinations ofany thereof.

In one embodiment the DFM may be selected from the followingBifidobacteria spp: Bifidobacterium lactis, Bifidobacterium bifidium,Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve,Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacteriumpseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacteriumangulatum, and combinations of any thereof.

Suitably the DFM may comprise a bacterium from one or more of thefollowing species: Bacillus subtilis, Bacillus licheniformis, Bacillusamyloliquefaciens, Enterococcus, Enterococcus spp, and Pediococcus spp,Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus,Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum,Bacillus subtilis, Propionibacterium thoenii, Lactobacillus farciminis,Lactobacillus rhamnosus, Megasphaera elsdenii, Clostridium butyricum,Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Bacilluscereus, Lactobacillus salivarius ssp. Salivarius, Propionibacteria spand combinations thereof.

The direct fed bacterium used in the present invention may be of thesame type (genus, species and strain) or may comprise a mixture ofgenera, species and/or strains.

Suitably the DFM according to the present invention may be one or moreof the products or the microorganisms contained in those products as inthe Table below:

Symbiotic Product Name Company Microorganism(s) ingredients EnvivaPro ®. Danisco A/S Bacillus subtilis strain 2084 (formerly known asAccession No. NRRl B-50013, Avicorr ®) Bacillus subtilis strain LSSAO1Accession No. NRRL B- 50104 and Bacillus subtilis strain 15A-P4 ATCCAccession No. PTA-6507 Calsporin ® Calpis - Japan Bacillus subtilisStrain C3102 Clostat ® Kemin Industries Bacillus subtilis Strain PB6Inc. Cylactin ® DSM Enterococcus NCIMB 10415 (SF68) Gallipro ® & Chr.Hansen A/S Bacillus subtilis Strain C3102 GalliproMax ® Gallipro ®Tect ®Chr. Hansen A/S Bacillus licheniformis Poultry star ® Biomin, IncEnterococcus and Pediococcus Fructo- oligosaccharides Protexin ®Protexin Int Lactobacillus, Bifidobacterium and another Proflora ®Alpharma Inc. Bacillus subtilis strain QST 713 β-Mos β-mannanoligosaccharides and β-glucans Ecobiol ® & Norel S.A. Bacillusamyloliquefaciens Ecobiol ® Plus CECT-5940 Fortiflora ® Enterococcusfaecium SF68 BioPlus2B ® DSM Bacillus subtilis and Bacilluslicheniformis Lactiferm ® Chr. Hansen Lactic acid bacteria 7Enterococcus faecium CSI ® Danisco A/S Bacillus strain Yea-Sacc ®Alltech Saccharomyces cerevisiae Biomin IMB52 ® Biomin EnterococcusBiomin C5 ® Biomin Pediococcus acidilactici, Enterococcus,Bifidobacterium animalis ssp. animalis, Lactobacillus reuteriLactobacillus salivarius ssp. salivarius Biacton ® ChemVet Lactobacillusfarciminis Oralin E1707 ® Chevita GmBH Enterococcus Probios-pioneer ChrHansen Enterococcus (2 strains) PDFM ® Lactococcus lactis DSM 11037Sorbiflore ® Danisco Animal Lactobacillus rhamnosus and NutritionLactobacillus farciminis Animavit ® KRKA Bacillus subtilis Bonvital ®Lactosan GmbH Enterococcus Levucell SB 20 ® Lallemand Saccharomycescerevisiae Levucell SC 0 & Lallemand Saccharomyces cerevisiae SC10 ® MEBactocell Lallemand Pediococcus acidilacti ActiSaf ® Le SaffreSaccharomyces cerevisiae (formerly BioSaf ®) Actisaf ® SC47 Le SaffreSaccharomyces cerevisiae NCYC Sc47 Miya-Gold ® Miyarisan Clostridiumbutyricum Pharma Fecinor and Norel S.A Enterococcus Fecinor Plus ®InteSwine ® ntegro Gidave Saccharomyces cerevisiae Ticaret AS NCYC R-625represented by R M Associates Ltd BioSprint ® ProSol SpA Saccharomycescerevisia Provita ® Provita Enterococcus and Lactobacillus rhamnosusPepSoyGen-C ® Regal BV Bacillus subtilis and (Nutraferma) Aspergillusoryzae Toyocerin ® Rubinum Bacillus cereus TOYOCERIN ® Rubinum Bacilluscereus var. toyoi NCIMB 40112/CNCM I-1012

In one embodiment suitably the DFM may be Enviva Pro®. Enviva Pro® iscommercially available from Danisco A/S and is a combination of Bacillusstrain 2084 Accession No. NRR1 B-50013, Bacillus strain LSSAO1 AccessionNo. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507(as taught in U.S. Pat. No. 7,754,469 B—incorporated herein byreference).

Suitably, the DFM may comprise a yeast from the genera: Saccharomycesspp.

Preferably the DFM to be used in accordance with the present inventionis a microorganism which is generally recognised as safe and, which ispreferably GRAS approved.

A skilled person will readily be aware of specific species and orstrains of microorganisms from within the genera described herein whichare used in the food and/or agricultural industries and which aregenerally considered suitable for animal consumption.

Preferably, the DFM used in accordance with the present invention is onewhich is suitable for animal consumption.

Advantageously, where the product is a feed or feed additivecomposition, the viable DFM should remain effective through the normal“sell-by” or “expiration” date of the product during which the feed orfeed additive composition is offered for sale by the retailer. Thedesired lengths of time and normal shelf life will vary from feedstuffto feedstuff and those of ordinary skill in the art will recognise thatshelf-life times will vary upon the type of feedstuff, the size of thefeedstuff, storage temperatures, processing conditions, packagingmaterial and packaging equipment.

In some embodiments it is important that the DFM is tolerant to heat,i.e. is thermotolerant. This is particularly the case where the feed ispelleted. Therefore in one embodiment the DFM may be a thermotolerantmicroorganism, such as a thermotolerant bacteria,_including for exampleBacillus spp.

In some embodiments it may be preferable that the DFM is a sporeproducing bacteria, such as Bacilli, e.g. Bacillus spp. Bacilli are ableto from stable endospores when conditions for growth are unfavorable andare very resistant to heat, pH, moisture and disinfectants.

In one embodiment suitably the DFM may decrease or prevent intestinalestablishment of pathogenic microorganism (such as Clostridiumperfringens and/or E. coli and/or Salmonella spp and/or Campylobacterspp.).

The DFM according to the present invention may be any suitable DFM. Inone embodiment the following assay “DFM ASSAY” may be used to determinethe suitability of a microorganism to be a DFM. For the avoidance ofdoubt in one embodiment a DFM selected as an inhibitory strain (or anantipathogen DFM) in accordance with the “DFM ASSAY” taught herein is asuitable DFM for use in accordance with the present invention, i.e. inthe feed additive composition according to the present invention.

DFM Assay:

Tubes were seeded each with a representative pathogen from arepresentative cluster.

Supernatant from a potential DFM grown aerobically or anaerobically wasadded to the seeded tubes and incubated.

After incubation, the optical density (OD) of the control andsupernatant treated tubes was measured for each pathogen.

Colonies of (potential DFM) strains that produced a lowered OD comparedwith the control were classified as an inhibitory strain (or anantipathogen DFM).

The DFM assay as used herein is explained in more detail inUS2009/0280090—incorporated herein by reference.

Preferably the representative pathogen used in assay is one (or more) ofthe following: Clostridium, such as Clostridium perfringens and/orClostridium difficile, and/or E. coli and/or Salmonella spp and/orCampylobacter spp. In one preferred embodiment the assay is conductedwith one or more of Clostridium perfringens and/or Clostridium difficileand/or E. coli, preferably Clostridium perfringens and/or Clostridiumdifficile, more preferably Clostridium perfringens.

In one embodiment the DFM of the present invention is preferably anantipathogen.

The term “antipathogen” as used herein means the DFM counters an effect(negative effect) of a pathogen.

In one embodiment to determine if a DFM is an antipathogenic DFM theabove mentioned DFM assay may be used. A DFM is considered to be anantipathogen or antipathogenic DFM if it is classed as an inhibitorystrain in the above mentioned “DFM assay”, for example when the pathogenis Clostridium perfringens.

In one embodiment the antipathogen DFM may be one or more of thefollowing bacteria:

Bacillus subtilis strain 2084 Accession No. NRRL B-50013,Bacillus subtilis strain LSSAO1 Accession No. NRRL B-50104,Bacillus subtilis strain 15A-P4 ATCC Accession No. PTA-6507,Bacillus subtilis strain 3A-P4 ATCC Accession No. PTA-6506, andBacillus subtilis strain BS27 ATCC Accession No. NRRL B-50105.

For the avoidance of doubt these strains are available and are referredto in U.S. Pat. No. 7,754,459 B.

In one embodiment the DFM used in accordance with the present inventionis not Lactobacillus gasseri BNR 17 Strain Acc No. KCTC 10902BP astaught in WO2008/016214.

Preferably the DFM is not an inactivated microorganism.

In one embodiment the DFM as used here is a composition comprising oneor more DFM microorganisms as described herein. The composition mayadditionally comprise the enzymes of the present invention. Thecomposition can be fed to an animal as a direct-fed microbial (DFM). Oneor more carrier(s) or other ingredients can be added to the DFM. The DFMmay be presented in various physical forms, for example, as a top dress,as a water soluble concentrate for use as a liquid drench or to be addedto a milk replacer, gelatin capsule, or gels. In one embodiment of thetop dress form, freeze-dried fermentation product is added to a carrier,such as whey, maltodextrin, sucrose, dextrose, limestone (calciumcarbonate), rice hulls, yeast culture, dried starch, and/or sodiumsilico aluminate. In one embodiment of the water soluble concentrate fora liquid drench or milk replacer supplement, freeze-dried fermentationproduct is added to a water soluble carrier, such as whey, maltodextrin,sucrose, dextrose, dried starch, sodium silico aluminate, and a liquidis added to form the drench or the supplement is added to milk or a milkreplacer. In one embodiment of the gelatin capsule form, freeze-driedfermentation product is added to a carrier, such as whey, maltodextrin,sugar, limestone (calcium carbonate), rice hulls, yeast culture driedstarch, and/or sodium silico aluminate. In one embodiment, the bacteriaand carrier are enclosed in a degradable gelatin capsule. In oneembodiment of the gels form, freeze-dried fermentation product is addedto a carrier, such as vegetable oil, sucrose, silicon dioxide,polysorbate 80, propylene glycol, butylated hydroxyanisole, citric acid,ethoxyquin, and/or artificial coloring to form the gel.

The DFM(s) may optionally be admixed with a dry formulation of additivesincluding but not limited to growth substrates, enzymes, sugars,carbohydrates, extracts and growth promoting micro-ingredients. Thesugars could include the following: lactose; maltose; dextrose;maltodextrin; glucose; fructose; mannose; tagatose; sorbose; raffinose;and galactose. The sugars range from 50-95%, either individually or incombination. The extracts could include yeast or dried yeastfermentation solubles ranging from 5-50%. The growth substrates couldinclude: trypticase, ranging from 5-25%; sodium lactate, ranging from5-30%; and, Tween 80, ranging from 1-5%. The carbohydrates could includemannitol, sorbitol, adonitol and arabitol. The carbohydrates range from5-50% individually or in combination. The micro-ingredients couldinclude the following: calcium carbonate, ranging from 0.5-5.0%; calciumchloride, ranging from 0.5-5.0%; dipotassium phosphate, ranging from0.5-5.0%; calcium phosphate, ranging from 0.5-5.0%; manganeseproteinate, ranging from 0.25-1.00%; and, manganese, ranging from0.25-1.0%.

To prepare DFMs described herein, the culture(s) and carrier(s) (whereused) can be added to a ribbon or paddle mixer and mixed for about 15minutes, although the timing can be increased or decreased. Thecomponents are blended such that a uniform mixture of the cultures andcarriers result. The final product is preferably a dry, flowable powder.The DFM(s) or composition comprising same can then be added to animalfeed or a feed premix, added to an animal's water, or administered inother ways known in the art (preferably simultaneously with the enzymesof the present invention). A feed for an animal can be supplemented withone or more DFM(s) described herein or with a composition describedherein.

By “a mixture of at least two strains,” is meant a mixture of two,three, four, five, six or even more strains. In some embodiments of amixture of strains, the proportions can vary from 1% to 99%. Otherembodiments of a mixture of strains are from 25% to 75%. Additionalembodiments of a mixture of strains are approximately 50% for eachstrain. When a mixture comprises more than two strains, the strains canbe present in substantially equal proportions or in differentproportions in the mixture.

The DFM may be dosed appropriately.

Suitably dosages of DFM in the feed may be between about 1×10³ CFU/gfeed to about 1×10⁹ CFU/g feed, suitably between about 1×10⁴ CFU/g feedto about 1×10⁸ CFU/g feed, suitably between about 7.5×10⁴ CFU/g feed toabout 1×10⁷ CFU/g feed.

In one embodiment the DFM is dosed in the feedstuff at more than about1×10³ CFU/g feed, suitably more than about 1×10⁴ CFU/g feed, suitablymore than about 7.5×10⁴ CFU/g feed.

Suitably dosages of DFM in the feed additive composition may be betweenabout 1×10⁵ CFU/g composition to about 1×10¹³ CFU/g composition,suitably between about 1×10⁶ CFU/g composition to about 1×10¹² CFU/gcomposition, suitably between about 3.75×10⁷ CFU/g composition to about1×10¹¹ CFU/g composition.

In one embodiment the DFM is dosed in the feed additive composition atmore than about 1×10⁵ CFU/g composition, suitably more than about 1×10⁶CFU/g composition, suitably more than about 3.75×10⁷ CFU/g composition.

In one embodiment the DFM is dosed in the feed additive composition atmore than about 2×10⁵ CFU/g composition, suitably more than about 2×10⁶CFU/g composition, suitably more than about 3.75×10⁷ CFU/g composition.

As used herein the term “CFU” means colony forming units and is ameasure of viable cells in which a colony represents an aggregate ofcells derived from a single progenitor cell.

Xylanase

Xylanase is the name given to a class of enzymes which degrade thelinear polysaccharide beta-1,4-xylan into xylose, thus breaking downhemicellulose, one of the major components of plant cell walls.

The xylanase for use in the present invention may be any commerciallyavailable xylanase.

Suitably the xylanase may be an endo-1,4-β-d-xylanase (classified asE.C. 3.2.1.8) or a 1,4,0-xylosidase (classified as E.C. 3.2.1.37).

In one embodiment preferably the xylanase in an endoxylanase, e.g. anendo-1,4-β-d-xylanase. The classification for an endo-1,4-β-d-xylanaseis E.C. 3.2.1.8.

In one embodiment the present invention relates to a DFM in combinationwith an endoxylanase, e.g. an endo-1,4-β-d-xylanase, and another enzyme.

All E.C. enzyme classifications referred to here relate to theclassifications provided in Enzyme Nomenclature—Recommendations (1992)of the nomenclature committee of the International Union of Biochemistryand Molecular Biology—ISBN 0-12-226164-3.

Suitably, the xylanase for use in the present invention may be axylanase from Bacillus, Trichoderma, Thermomyces, Aspergillus andPenicillium.

In one embodiment the xylanase may be the xylanase in Axtra XAP® orAvizyme 1502®, both commercially available products from Danisco A/S.

In one preferred embodiment the xylanase for use in the presentinvention may be one or more of the xylanases in one or more of thecommercial products below:

Commercial Name ® Company Xylanase type Xylanase source Allzyme PTAlltech endo-1,4-β-xylanase Aspergillus Niger Amylofeed Andrésendo-1,4-β-xylanase Aspergillus Niger (phoenicis) Pintaluba S.A Avemix02 CS Aveve endo-1,4-β-xylanase Trichoderma reesei AveMix XG 10 Aveve,NL endo-1,4-β-xylanase Trichoderma reesei Avizyme 1100 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme 1110 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme 1202 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme 1210 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme 1302 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme 1500 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme 1505 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme SX Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Belfeed MP100 Beldemendo-1,4-β-xylanase Bacillus subtilis Biofeed Plus DSMendo-1,4-β-xylanase Humicola insolens Danisco Glycosidase Danisco Animalendo-1,4-β-xylanase Trichoderma reesei (TPT/L) Nutrition DaniscoXylanase Danisco endo-1,4-β-xylanase Trichoderma reesei Econase XTABVista endo-1,4-β-xylanase Trichoderma reesei Endofeed ® DC Andresendo-1,4-β-xylanase Aspergillus Niger Pintaluba S.A. Feedlyve AXL Lyvenendo-1,4-β-xylanase Trichoderma longibrachiatum Grindazym GP Daniscoendo-1,4-β-xylanase Aspergillus Niger Grindazym GV Daniscoendo-1,4-β-xylanase Aspergillus Niger Hostazym X Huvepharmaendo-1,4-β-xylanase Trichoderma longibrachiatum Kemzyme Plus Dry Keminendo-1,4-β-xylanase Trichoderma viride Kemzyme Plus Liquid Keminendo-1,4-β-xylanase Trichoderma viride Kemzyme W dry Keminendo-1,4-β-xylanase Trichoderma viride Kemzyme W liquid Keminendo-1,4-β-xylanase Trichoderma viride Natugrain BASFendo-1,4-β-xylanase Trichoderma longibrachiatum Natugrain TS Plus BASFendo-1,4-β-xylanase Aspergillus Niger Natugrain Wheat BASFendo-1,4-β-xylanase Aspergillus Niger Natugrain ® TS/L BASFendo-1,4-β-xylanase Aspergillus Niger Natuzyme Bioprotonendo-1,4-β-xylanase Trichoderma longibrachiatum/ Trichoderma reeseiPorzyme 8100 Danisco endo-1,4-β-xylanase Trichoderma longibrachiatumPorzyme 8300 Danisco endo-1,4-β-xylanase Trichoderma longibrachiatumPorzyme 9102 Danisco endo-1,4-β-xylanase Trichoderma longibrachiatumPorzyme 9310/ Danisco endo-1,4-β-xylanase Trichoderma longibrachiatumAvizyme 1310 Porzyme tp100 Danisco endo-1,4-β-xylanase Trichodermalongibrachiatum Ronozyme AX DSM endo-1,4-β-xylanase Thermomyceslanuginosus gene expressed in Aspergillus oryzae Ronozyme WXDSM/Novozymes endo-1,4-β-xylanase Thermomyces lanuginosus gene expressedin Aspergillus oryzae Rovabio Excel Adisseo endo-1,4-β-xylanasePenicillium funiculosum Roxazyme G2 DSM/Novozymes endo-1,4-β-xylanaseTrichoderma longibrachiatum Safizym X Le Saffre endo-1,4-β-xylanaseTrichoderma longibrachiatum Xylanase Lyven endo-1,4-β-xylanaseTrichoderma longibrachiatum

Preferably, the xylanase is present in the feedstuff in range of about500 XU/kg to about 16,000 XU/kg feed, more preferably about 750 XU/kgfeed to about 8000 XU/kg feed, and even more preferably about 1000 XU/kgfeed to about 4000 XU/kg feed

In one embodiment the xylanase is present in the feedstuff at more thanabout 500 XU/kg feed, suitably more than about 600 XU/kg feed, suitablymore than about 700 XU/kg feed, suitably more than about 800 XU/kg feed,suitably more than about 900 XU/kg feed, suitably more than about 1000XU/kg feed.

In one embodiment the xylanase is present in the feedstuff at less thanabout 16,000 XU/kg feed, suitably less than about 8000 XU/kg feed,suitably less than about 7000 XU/kg feed, suitably less than about 6000XU/kg feed, suitably less than about 5000 XU/kg feed, suitably less thanabout 4000 XU/kg feed.

Preferably, the xylanase is present in the feed additive composition inrange of about 100 XU/g to about 320,000 XU/g composition, morepreferably about 300 XU/g composition to about 160,000 XU/g composition,and even more preferably about 500 XU/g composition to about 50,000 XU/gcomposition, and even more preferably about 500 XU/g composition toabout 40,000 XU/g composition.

In one embodiment the xylanase is present in the feed additivecomposition at more than about 100 XU/g composition, suitably more thanabout 200 XU/g composition, suitably more than about 300 XU/gcomposition, suitably more than about 400 XU/g composition, suitablymore than about 500 XU/g composition.

In one embodiment the xylanase is present in the feed additivecomposition at less than about 320,000 XU/g composition, suitably lessthan about 160,000 XU/g composition, suitably less than about 50,000XU/g composition, suitably less than about 40,000 XU/g composition,suitably less than about 30000 XU/g composition.

It will be understood that one xylanase unit (XU) is the amount ofenzyme that releases 0.5 μmol of reducing sugar equivalents (as xyloseby the Dinitrosalicylic acid (DNS) assay-reducing sugar method) from aoat-spelt-xylan substrate per min at pH 5.3 and 50° C. (Bailey, M. J.Biely, P. and Poutanen, K., Journal of Biotechnology, Volume 23, (3),May 1992, 257-270).

In one embodiment suitably the enzyme is classified using the E.C.classification above, and the E.C. classification designates an enzymehaving that activity when tested in the assay taught herein fordetermining 1 XU.

Amylase

Amylase is the name given to a class of enzymes capable of hydrolysingstarch to shorter-chain oligosaccharides such as maltose. The glucosemoiety can then be more easily transferred from maltose to amonoglyceride or glycosylmonoglyceride than from the original starchmolecule.

The term amylase includes α-amylases (E.C. 3.2.1.1), G4-forming amylases(E.C. 3.2.1.60), β-amylases (E.C. 3.2.1.2) and 7-amylases (E.C.3.2.1.3).

In one embodiment preferably the amylase is an α-amylase. α-Amylases areclassified as (E.C. 3.2.1.1).

These can include amylases of bacterial or fungal origin, chemicallymodified or protein engineered mutants are included.

In one embodiment preferably the amylase may be an amylase, e.g. anα-amylase, from Bacillus licheniformis and/or an amylase, e.g. anα-amylase, from Bacillus amyloliquefaciens.

In one embodiment the α-amylase may be the α-amylase in Axtra XAP® orAvizyme 1502®, both commercially available products from Danisco A/S.

In another embodiment the amylase may be a pepsin resistant α-amylase,such as a pepsin resistant Trichoderma (such as Trichoderma reesei)alpha amylase. A suitably pepsin resistant α-amylase is taught in UKapplication number 1011513.7 (which is incorporated herein by reference)and PCT/IB2011/053018 (which is incorporated herein by reference).

In one embodiment the amylase may be a pepsin resistant α-amylasecomprising or consisting of an amino acid sequence:

-   -   i) as set forth in SEQ ID No. 1 or SEQ ID No. 3;    -   ii) as set forth in SEQ ID No. 1 or SEQ ID No. 3 except for one        or several amino acid additions/insertions, deletions or        substitutions;    -   iii) having at least 85% (preferably, at least 90%, 95%, 97%,        98% or 99%) identity to SEQ ID No. 1 or at least 70%        (preferably, at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%)        identity to SEQ ID No. 3;    -   iv) which is produced by expression of a nucleotide sequence        comprising the sequence of SEQ ID No. 2 or SEQ ID No. 4;    -   v) which is produced by expression of a nucleotide sequence        which differs from SEQ ID No. 2 or SEQ ID No. 4 due to the        degeneracy of the genetic code;    -   vi) which is produced by expression of a nucleotide sequence        which differs from SEQ ID No. 2 or SEQ ID No. 4 by one or        several nucleotide additions/insertions, deletions or        substitutions; or    -   vii) which is produced by expression of a nucleotide sequence        which has at least 70% (preferably, at least 75%, 80%, 85%, 90%,        95%, 97%, 98% or 99%) identity to SEQ ID No. 2 or SEQ ID No. 4.

The pepsin resistant alpha amylase may also be encoded by a nucleotidesequence which hybridises to SEQ ID No. 2 or SEQ ID No. 4 understringent or highly stringent conditions.

In one preferred embodiment the amylase for use in the present inventionmay be one or more of the amylases in one or more of the commercialproducts below:

Commercial product ® Company Amylase type Amylase source AmylofeedAndrés alpha amylase Aspergillus oryzae Pintaluba S.A Avizyme 1500Danisco alpha amylase Bacillus amyloliquefaciens Avizyme 1505 Daniscoalpha amylase Bacillus amyloliquefaciens Kemzyme Plus Dry Keminalpha-amylase Bacillus amyloliquefaciens Kemzyme Plus Liquid Keminalpha-amylase Bacillus amyloliquefaciens Kemzyme W dry Keminalpha-amylase Bacillus amyloliquefaciens Kemzyme W liquid Keminalpha-amylase Bacillus amyloliquefaciens Natuzyme Bioprotonalpha-amylase Trichoderma longibrachiatum/ Trichoderma reesei Porzyme8100 Danisco alpha-amylase Bacillus amyloliquefaciens Porzyme tp100Danisco alpha-amylase Bacillus amyloliquefaciens Ronozyme ADSM/Novozymes alpha-amylase Bacillus amyloliquefaciens Ronozyme AX DSMalpha-amylase Bacillus amyloliquefaciens Ronozyme ® RumiStarDSM/Novozymes alpha-amylase Bacillus stearothermophilus (L/CT) expressedin Bacillus licheniformis

In one embodiment the amylase may be a maltogenic alpha-amylase fromBacillus (see EP 120 693). This amylase is commercially available underthe trade name Novamyl (Novo Nordisk A/S, Denmark). Novamyl is describedin detail in International Patent Publication WO 91/104669.

Preferably, the amylase is present in the feedstuff in range of about 50AU/kg to about 10,000 AU/kg feed, more preferably about 70 AU/kg feed toabout 7500 AU/kg feed, more preferably about 70 AU/kg feed to about 5000AU/kg feed and even more preferably about 100 AU/kg feed to about 2000AU/kg feed.

In one embodiment the amylase is present in the feedstuff at more thanabout 50 AU/kg feed, suitably more than about 60 AU/kg feed, suitablymore than about 70 AU/kg feed, suitably more than about 80 AU/kg feed,suitably more than about 90 AU/kg feed, suitably more than about 100AU/kg feed.

In one embodiment the amylase is present in the feedstuff at less thanabout 10,000 AU/kg feed, suitably less than about 8000 AU/kg feed,suitably less than about 7000 AU/kg feed, suitably less than about 5000AU/kg feed, suitably less than about 4000 AU/kg feed, suitably less thanabout 3000 AU/kg feed, suitably less than about 2000 AU/kg feed.

Preferably, the amylase is present in the feed additive composition inrange of about 10 AU/kg to about 200,000 AU/g composition, morepreferably about 30 AU/g composition to about 100,000 AU/g composition,and even more preferably about 40 AU/g composition to about 50,000 AU/gcomposition, and even more preferably about 50 AU/g composition to about20,000 AU/g composition.

In one embodiment the amylase is present in the feed additivecomposition at more than about 10 AU/g composition, suitably more thanabout 20 AU/g composition, suitably more than about 30 AU/g composition,suitably more than about 40 AU/g composition, suitably more than about50 AU/g composition.

In one embodiment the amylase is present in the feed additivecomposition at less than about 200,000 AU/g composition, suitably lessthan about 100,000 AU/g composition, suitably less than about 50,000AU/g composition, suitably less than about 40,000 AU/g composition,suitably less than about 30000 AU/g composition, suitably less thanabout 20000 AU/g composition.

It will be understood that one amylase unit (AU) is the amount of enzymethat releases 1 mmol of glucosidic linkages from a water insolublecross-linked starch polymer substrate per min at pH 6.5 and 37° C. (thismay be referred to herein as the assay for determining 1 AU).

1 TAU (α-amylase activity) is the amount of enzyme required to release(in the presence of excess α-glucosidase) 0.20 mmol of glucosidiclinkages (expressed as p-nitrophenol equivalents) from a maltoheptaosidesubstrate per minute at pH 8.0 and 40° C. This may be referred to hereinas the assay for determining 1 TAU unit.

In one embodiment suitably the enzyme is classified using the E.C.classification above, and the E.C. classification designates an enzymehaving that activity when tested in the assay taught herein fordetermining 1 AU.

Protease

The term protease as used herein is synonymous with peptidase orproteinase.

The protease for use in the present invention may be a subtilisin (E.C.3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serineprotease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x).

Preferably the protease in accordance with the present invention is asubtilisin.

Suitable proteases include those of animal, vegetable or microbialorigin. Chemically modified or protein engineered mutants are alsosuitable. The protease may be a serine protease or a metalloprotease,e.g., an alkaline microbial protease or a trypsin-like protease.Examples of alkaline proteases are subtilisins, especially those derivedfrom Bacillus sp., e.g., subtilisin Novo, subtilisin Carlsberg,subtilisin 309 (see, e.g., U.S. Pat. No. 6,287,841), subtilisin 147, andsubtilisin 168 (see, e.g., WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin), and Fusariumproteases (see, e.g., WO 89/06270 and WO 94/25583). Examples of usefulproteases also include but are not limited to the variants described inWO 92/19729 and WO 98/20115.

In one preferred embodiment the protease for use in the presentinvention may be one or more of the proteases in one or more of thecommercial products below:

Commercial product ® Company Protease type Protease source Avizyme 1100Danisco A/S Subtilisin Bacillus subtilis Avizyme 1202 Danisco A/SSubtilisin Bacillus subtilis Avizyme 1302 Danisco A/S SubtilisinBacillus subtilis Avizyme 1500 Danisco A/S Subtilisin Bacillus subtilisAvizyme 1505 Danisco A/S Subtilisin Bacillus subtilis Kemzyme Plus DryKemin Bacillolysin Bacillus amyloliquefaciens Kemzyme W dry KeminBacillolysin Bacillus amyloliquefaciens Natuzyme Bioproton ProteaseTrichoderma longibrachiatum/ Trichoderma reesei Porzyme 8300 DaniscoSubtilisin Bacillus subtilis Ronozyme ProAct DSM/Novozymes Alkalineserine Nocardiopsis prasina protease gene expressed in Bacilluslicheniformis Versazyme/Cibenza Novus Keratinase Bacillus licheniformisDP100

In one embodiment the protease may be a protease from B. subtilis.

In one embodiment the protease may be a Nocardiopsis protease availablefrom Novozymes A/S.

Preferably, the protease is present in the feedstuff in range of about1000 U/kg to about 20,000 PU/kg feed, more preferably about 1500 PU/kgfeed to about 10000 PU/kg feed, more preferably about 2000 PU/kg feed toabout 6000 PU/kg feed.

In one embodiment the protease is present in the feedstuff at more thanabout 1000 PU/kg feed, suitably more than about 1500 PU/kg feed,suitably more than about 2000 PU/kg feed.

In one embodiment the protease is present in the feedstuff at less thanabout 20,000 PU/kg feed, suitably less than about 10000 PU/kg feed,suitably less than about 7000 PU/kg feed, suitably less than about 6000PU/kg feed.

Preferably, the protease is present in the feed additive composition inrange of about 200 PU/g to about 400,000 PU/g composition, morepreferably about 300 PU/g composition to about 200,000 PU/g composition,and even more preferably about 5000 PU/g composition to about 100,000PU/g composition, and even more preferably about 700 PU/g composition toabout 70,000 PU/g composition, and even more preferably about 1000 PU/gcomposition to about 60,000 PU/g composition.

In one embodiment the protease is present in the feed additivecomposition at more than about 200 PU/g composition, suitably more thanabout 300 PU/g composition, suitably more than about 400 PU/gcomposition, suitably more than about 500 PU/g composition, suitablymore than about 750 PU/g composition, suitably more than about 1000 PU/gcomposition.

In one embodiment the protease is present in the feed additivecomposition at less than about 400,000 PU/g composition, suitably lessthan about 200,000 PU/g composition, suitably less than about 100,000PU/g composition, suitably less than about 80,000 PU/g composition,suitably less than about 70000 PU/g composition, suitably less thanabout 60000 PU/g composition.

It will be understood that one protease unit (PU) is the amount ofenzyme that liberates from the substrate (0.6% casein solution) onemicrogram of phenolic compound (expressed as tyrosine equivalents) inone minute at pH 7.5 (40 mM Na₂PO₄/lactic acid buffer) and 40° C. Thismay be referred to as the assay for determining 1 PU.

In one embodiment suitably the enzyme is classified using the E.C.classification above, and the E.C. classification designates an enzymehaving that activity when tested in the assay taught herein fordetermining 1 PU.

Phytase

The phytase for use in the present invention may be classified a6-phytase (classified as E.C. 3.1.3.26) or a 3-phytase (classified asE.C. 3.1.3.8).

In one embodiment the phytase may be a 6-phytase (E.C. 3.1.3.26).

In one preferred embodiment the phytase for use in the present inventionmay be one or more of the phytases in one or more of the commercialproducts below:

Commercial product ® Company Phytase type Phytase source Finase ABVista3-phytase Trichoderma reesei Finase EC ABVista 6-phytase E. coli geneexpressed in Trichoderma reesei Natuphos BASF 3-phytase AspergillusNiger Natuzyme Bioproton phytase (type Trichoderma longibrachiatum/ notspecified) Trichoderma reesei OPTIPHOS ® Huvepharma AD 6-phytase E. coligene expressed in Pichia pastoris Phytase sp1002 DSM 3-phytase Aconsensus gene expressed in Hansenula polymorpha Phyzyme XP Danisco6-phytase E. coli gene expressed in Schizosaccahomyces pombe Quantum2500D, 5000L ABVista 6-phytase E. coli gene expressed in Pichia pastorisor Trichoderma Ronozyme Hi-Phos DSM/Novozymes 6-phytase Citrobacterbraakii (M/L) gene expressed in Aspergillus oryzae Ronozyme NPDSM/Novozymes 6-phytase Peniphora lycii gene expressed in Aspergillusoryzae Ronozyme P DSM/Novozymes 6-phytase Peniphora lycii gene expressedin Aspergillus oryzae Rovabio PHY Adisseo 3-phytase Penicilliumfuniculosum

The term consensus gene as used herein means that the DNA vector used totransform the organism contains a synthetic phytase gene based on aconsensus sequence, a URA gene from the non-pathogenic yeastSaccharomyces cerevisiae and the origin of replication of theEscherichia coli plasmid pBR322.

In one embodiment the phytase is a Citrobacter phytase derived from e.g.Citrobacter freundii, preferably C. freundii NCIMB 41247 and variantsthereof e.g. as disclosed in WO2006/038062 (incorporated herein byreference) and WO2006/038128 (incorporated herein by reference),Citrobacter braakii YH-15 as disclosed in WO 2004/085638, Citrobacterbraakii ATCC 51113 as disclosed in WO2006/037328 (incorporated herein byreference), as well as variants thereof e.g. as disclosed inWO2007/112739 (incorporated herein by reference) and WO2011/117396(incorporated herein by reference), Citrobacter amalonaticus, preferablyCitrobacter amalonaticus ATCC 25405 or Citrobacter amalonaticus ATCC25407 as disclosed in WO2006037327 (incorporated herein by reference),Citrobacter gillenii, preferably Citrobacter gillenii DSM 13694 asdisclosed in WO2006037327 (incorporated herein by reference), orCitrobacter intermedius, Citrobacter koseri, Citrobacter murliniae,Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii,Citrobacter youngae, Citrobacter species polypeptides or variantsthereof.

In one embodiment the phytase may be a phytase from Citrobacter, e.g.from Citrobacter freundii, such as the phytase enzyme(s) taught inWO2006/038128, which reference is incorporated herein by reference.

In preferred embodiments, the phytase is preferably E. coli phytasemarketed under the name Phyzyme XP™ by Danisco A/S.

Alternatively the phytase may be a Buttiauxella phytase, e.g. aButtiauxella agrestis phytase, for example, the phytase enzymes taughtin WO 2006/043178, WO 2008/097619, WO2009/129489, WO2008/092901,PCT/U52009/41011 or PCTAB2010/051804, all of which are incorporatedherein by reference.

In one embodiment the phytase may be a phytase from Hafnia, e.g. fromHafnia alvei, such as the phytase enzyme(s) taught in US2008263688,which reference is incorporated herein by reference.

In one embodiment the phytase may be a phytase from Aspergillus, e.g.from Apergillus orzyae.

In one embodiment the phytase may be a phytase from Penicillium, e.g.from Penicillium funiculosum.

Preferably, the phytase is present in the feedstuff in range of about200 FTU/kg to about 1000 FTU/kg feed, more preferably about 300 FTU/kgfeed to about 750 FTU/kg feed, more preferably about 400 FTU/kg feed toabout 500 FTU/kg feed.

In one embodiment the phytase is present in the feedstuff at more thanabout 200 FTU/kg feed, suitably more than about 300 FTU/kg feed,suitably more than about 400 FTU/kg feed.

In one embodiment the phytase is present in the feedstuff at less thanabout 1000 FTU/kg feed, suitably less than about 750 FTU/kg feed.

Preferably, the phytase is present in the feed additive composition inrange of about 40 FTU/g to about 40,000 FTU/g composition, morepreferably about 80 FTU/g composition to about 20,000 FTU/g composition,and even more preferably about 100 FTU/g composition to about 10,000FTU/g composition, and even more preferably about 200 FTU/g compositionto about 10,000 FTU/g composition.

In one embodiment the phytase is present in the feed additivecomposition at more than about 40 FTU/g composition, suitably more thanabout 60 FTU/g composition, suitably more than about 100 FTU/gcomposition, suitably more than about 150 FTU/g composition, suitablymore than about 200 FTU/g composition.

In one embodiment the phytase is present in the feed additivecomposition at less than about 40,000 FTU/g composition, suitably lessthan about 20,000 FTU/g composition, suitably less than about 15,000FTU/g composition, suitably less than about 10,000 FTU/g composition.

It will be understood that as used herein 1 FTU (phytase unit) isdefined as the amount of enzyme required to release 1 μmol of inorganicorthophosphate from a substrate in one minute under the reactionconditions defined in the ISO 2009 phytase assay—A standard assay fordetermining phytase activity and 1 FTU can be found at InternationalStandard ISO/DIS 30024: 1-17, 2009.

In one embodiment suitably the enzyme is classified using the E.C.classification above, and the E.C. classification designates an enzymehaving that activity when tested in the assay taught herein fordetermining 1 FTU.

Advantages

The interaction of DFMs with enzymes is complicated and without wishingto be bound by theory, it is very surprising that we can see animprovement in the subject's resistance to necrotic enteritis, e.g. thatwe see a reduction in lesion scores for instance. Prior to the presentinvention the combination of DFMs and enzymes (e.g. as taught herein)had not been taught for this specific purpose.

One advantage of the present invention is that the feed additivecomposition according to the present invention can avoid the negativeeffects of necrotic enteritis or can be used for improving the subject'sresistance to necrotic enteritis.

Without wishing to be bound by theory, phytase catalyses the sequentialhydrolysis of phytate, a principal storage form of phosphorus in cerealsand legumes, to less phosphorylated myo-inositol derivatives withconcomitant release of inorganic phosphate. Hydrolysis of phytate causesa reduction of endogenous losses of amino acids to the intestinal lumen.A reduction of endogenous amino acid losses in the intestine reduces theavailability of nitrogen for bacterial growth, which helps the activityof DFMs on inhibition of C. perfringens and other pathogenic bacteria.

Without wishing to be bound in theory proteases cause non-specifichydrolysis of dietary protein yielding a variety of polypeptides in theintestinal lumen. Animals finalise protein hydrolysis and absorb suchamino acids. However, in the case of enteric pathogenic challenges,pathogenic bacteria may take advantage of higher peptide availability inthe lumen of jejunum and ileum. DFMs inhibit the growth ofentero-pathogens by for example competing for N sources, as well as bydirect inhibition.

In addition, xylanase degrades the linear polysaccharide beta-1,4-xylaninto xylose. Without wishing to be bound by theory, the inventors hereinhave shown that the increased energy digestibility with the combinationof DFMs and enzymes is not explained by starch, fat or protein,therefore it must be explained by non-starch polysaccharides.

Amylase activity hydrolyses alpha-bonds of large alpha-linkedpolysaccharides such as starch yielding dextrins and oligosaccharides,which are mainly absorbed in the small intestine after hydrolysis tomaltose and glucose in the gut wall. Surprisingly, rapid starchhydrolysis in the foregut and greater absorption of glucose in theduodenum deprives pathogenic bacteria from an important energy source(glucose) in the jejunum and ileum, which improves the DFM activitybecause of a competitive advantage against pathogens that cannot usepentoses as efficiently.

In combination the four enzymes and DFMs surprisingly provide asignificant improvement on the pathogen reduction and/or resistance tonecrotic enteritis compared with other DFM and enzyme combinationsand/or DFMs alone and/or enzyme(s) alone.

The specific combination of DFMs and the enzymes taught herein mayadvantageously lead to reduced mucin secretion. Without wishing to bebound by theory this reduced mucin secretion may result in a reductionof endogenous amino acid losses, and/or may be responsible for improvedperformance.

The specific combination of DFMs and the enzymes taught herein mayadvantageously reduce inflammation in the ileum. This can be seen by thedownregulation of IFR-g expression in the ileum. The inventors haveshown that modulation of immune response may improve performance.

Formulation of the DFM with the Enzymes

The DFM and the enzymes may be formulated in any suitable way to ensurethat the formulation comprises viable DFMs and active enzymes.

In one embodiment the DFM and enzymes may be formulated as a liquid, adry powder or a granule.

The dry powder or granules may be prepared by means known to thoseskilled in the art, such as, in top-spray fluid bed coater, in a buttomspray Wurster or by drum granulation (e.g. High sheer granulation),extrusion, pan coating or in a microingredients mixer.

For some embodiments the DFM and/or the enzyme(s) may be coated, forexample encapsulated. Suitably the DFM and enzymes may be formulatedwithin the same coating or encapsulated within the same capsule.Alternatively one or two or three or four of the enzymes may beformulated within the same coating or encapsulated within the samecapsule and the DFM could be formulated in a coating separate to the oneor more or all of the enzymes. In some embodiments, such as where theDFM is capable of producing endospores, the DFM may be provided withoutany coating. In such circumstances, the DFM endospores may be simplyadmixed with one or two or three or four enzymes. In the latter case,the enzymes may be coated, e.g. encapsulated, for instance one or moreor all of the enzymes may be coated, e.g. encapsulated. The enzymes maybe encapsulated as mixtures (i.e. comprising one or more, two or more,three or more or all) of enzymes or they may be encapsulated separately,e.g. as single enzymes. In one preferred embodiment all four enzymes maybe coated, e.g. encapsulated, together.

In one embodiment the coating protects the enzymes from heat and may beconsidered a thermoprotectant.

In one embodiment the feed additive composition is formulated to a drypowder or granules as described in WO2007/044968 (referred to as TPTgranules) or WO1997/016076 or WO1992/012645 (each of which isincorporated herein by reference).

In one embodiment the feed additive composition may be formulated to agranule for feed compositions comprising: a core; an active agent; andat least one coating, the active agent of the granule retaining at least50% activity, at least 60% activity, at least 70% activity, at least 80%activity after conditions selected from one or more of a) a feedpelleting process, b) steam-heated feed pretreatment process, c)storage, d) storage as an ingredient in an unpelleted mixture, and e)storage as an ingredient in a feed base mix or a feed premix comprisingat least one compound selected from trace minerals, organic acids,reducing sugars, vitamins, choline chloride, and compounds which resultin an acidic or a basic feed base mix or feed premix.

With regard to the granule at least one coating may comprise a moisturehydrating material that constitutes at least 55% w/w of the granule;and/or at least one coating may comprise two coatings. The two coatingsmay be a moisture hydrating coating and a moisture barrier coating. Insome embodiments, the moisture hydrating coating may be between 25% and60% w/w of the granule and the moisture barrier coating may be between2% and 15% w/w of the granule. The moisture hydrating coating may beselected from inorganic salts, sucrose, starch, and maltodextrin and themoisture barrier coating may be selected from polymers, gums, whey andstarch.

The granule may be produced using a feed pelleting process and the feedpretreatment process may be conducted between 70° C. and 95° C. for upto several minutes, such as between 85° C. and 95° C.

In one embodiment the feed additive composition may be formulated to agranule for animal feed comprising: a core; an active agent, the activeagent of the granule retaining at least 80% activity after storage andafter a steam-heated pelleting process where the granule is aningredient; a moisture barrier coating; and a moisture hydrating coatingthat is at least 25% w/w of the granule, the granule having a wateractivity of less than 0.5 prior to the steam-heated pelleting process.

The granule may have a moisture barrier coating selected from polymersand gums and the moisture hydrating material may be an inorganic salt.The moisture hydrating coating may be between 25% and 45% w/w of thegranule and the moisture barrier coating may be between 2% and 10% w/wof the granule.

The granule may be produced using a steam-heated pelleting process whichmay be conducted between 85° C. and 95° C., for up to several minutes.

In some embodiments the DFM (e.g. DFM endospores for example) may bediluted using a diluent, such as starch powder, lime stone or the like.

In one embodiment, the composition is in a liquid formulation suitablefor consumption preferably such liquid consumption contains one or moreof the following: a buffer, salt, sorbitol and/or glycerol.

In another embodiment the feed additive composition may be formulated byapplying, e.g. spraying, the enzyme(s) onto a carrier substrate, such asground wheat for example.

In one embodiment the feed additive composition according to the presentinvention may be formulated as a premix. By way of example only thepremix may comprise one or more feed components, such as one or moreminerals and/or one or more vitamins.

In one embodiment the DFM and/or enzymes for use in the presentinvention are formulated with at least one physiologically acceptablecarrier selected from at least one of maltodextrin, limestone (calciumcarbonate), cyclodextrin, wheat or a wheat component, sucrose, starch,Na₂SO₄, Talc, PVA, sorbitol, benzoate, sorbiate, glycerol, sucrose,propylene glycol, 1,3-propane diol, glucose, parabens, sodium chloride,citrate, acetate, phosphate, calcium, metabisulfite, formate andmixtures thereof.

Packaging

In one embodiment the feed additive composition and/or premix and/orfeed or feedstuff according to the present invention is packaged.

In one preferred embodiment the feed additive composition and/or premixand/or feed or feedstuff is packaged in a bag, such as a paper bag.

In an alternative embodiment the feed additive composition and/or premixand/or feed or feedstuff may be sealed in a container. Any suitablecontainer may be used.

Feed

The feed additive composition of the present invention may be used as—orin the preparation of—a feed.

The term “feed” is used synonymously herein with “feedstuff”.

The feed may be in the form of a solution or as a solid—depending on theuse and/or the mode of application and/or the mode of administration.

When used as—or in the preparation of—a feed—such as functional feed—thecomposition of the present invention may be used in conjunction with oneor more of: a nutritionally acceptable carrier, a nutritionallyacceptable diluent, a nutritionally acceptable excipient, anutritionally acceptable adjuvant, a nutritionally active ingredient.

In a preferred embodiment the feed additive composition of the presentinvention is admixed with a feed component to form a feedstuff.

The term “feed component” as used herein means all or part of thefeedstuff. Part of the feedstuff may mean one constituent of thefeedstuff or more than one constituent of the feedstuff, e.g. 2 or 3 or4. In one embodiment the term “feed component” encompasses a premix orpremix constituents.

Preferably the feed may be a fodder, or a premix thereof, a compoundfeed, or a premix thereof. In one embodiment the feed additivecomposition according to the present invention may be admixed with acompound feed, a compound feed component or to a premix of a compoundfeed or to a fodder, a fodder component, or a premix of a fodder.

The term fodder as used herein means any food which is provided to ananimal (rather than the animal having to forage for it themselves).Fodder encompasses plants that have been cut.

The term fodder includes hay, straw, silage, compressed and pelletedfeeds, oils and mixed rations, and also sprouted grains and legumes.

Fodder may be obtained from one or more of the plants selected from:alfalfa (lucerne), barley, birdsfoot trefoil, brassicas, Chau moellier,kale, rapeseed (canola), rutabaga (swede), turnip, clover, alsikeclover, red clover, subterranean clover, white clover, grass, false oatgrass, fescue, Bermuda grass, brome, heath grass, meadow grasses (fromnaturally mixed grassland swards, orchard grass, rye grass,Timothy-grass, corn (maize), millet, oats, sorghum, soybeans, trees(pollard tree shoots for tree-hay), wheat, and legumes.

The term “compound feed” means a commercial feed in the form of a meal,a pellet, nuts, cake or a crumble. Compound feeds may be blended fromvarious raw materials and additives. These blends are formulatedaccording to the specific requirements of the target animal.

Compound feeds can be complete feeds that provide all the daily requirednutrients, concentrates that provide a part of the ration (protein,energy) or supplements that only provide additional micronutrients, suchas minerals and vitamins.

The main ingredients used in compound feed are the feed grains, whichinclude corn, soybeans, sorghum, oats, and barley.

Suitably a premix as referred to herein may be a composition composed ofmicroingredients such as vitamins, minerals, chemical preservatives,antibiotics, fermentation products, and other essential ingredients.Premixes are usually compositions suitable for blending into commercialrations.

Any feedstuff of the present invention may comprise one or more feedmaterials selected from the group comprising a) cereals, such as smallgrains (e.g., wheat, barley, rye, oats and combinations thereof) and/orlarge grains such as maize or sorghum; b) by products from cereals, suchas corn gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran,wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palmkernel, and citrus pulp; c) protein obtained from sources such as soya,sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal,dried plasma protein, meat and bone meal, potato protein, whey, copra,sesame; d) oils and fats obtained from vegetable and animal sources; e)minerals and vitamins.

A feedstuff of the present invention may contain at least 30%, at least40%, at least 50% or at least 60% by weight corn and soybean meal orcorn and full fat soy, or wheat meal or sunflower meal.

In addition or in the alternative, a feedstuff of the present inventionmay comprise at least one high fibre feed material and/or at least oneby-product of the at least one high fibre feed material to provide ahigh fibre feedstuff. Examples of high fibre feed materials include:wheat, barley, rye, oats, by products from cereals, such as corn glutenmeal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheatmiddlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel,and citrus pulp. Some protein sources may also be regarded as highfibre: protein obtained from sources such as sunflower, lupin, favabeans and cotton.

In the present invention the feed may be one or more of the following: acompound feed and premix, including pellets, nuts or (cattle) cake; acrop or crop residue: corn, soybeans, sorghum, oats, barley, cornstover, copra, straw, chaff, sugar beet waste; fish meal; freshly cutgrass and other forage plants; meat and bone meal; molasses; oil cakeand press cake; oligosaccharides; conserved forage plants: hay andsilage; seaweed; seeds and grains, either whole or prepared by crushing,milling etc.; sprouted grains and legumes; yeast extract.

The term feed in the present invention also encompasses in someembodiments pet food. A pet food is plant or animal material intendedfor consumption by pets, such as dog food or cat food. Pet food, such asdog and cat food, may be either in a dry form, such as kibble for dogs,or wet canned form. Cat food may contain the amino acid taurine.

The term feed in the present invention also encompasses in someembodiments fish food. A fish food normally contains macro nutrients,trace elements and vitamins necessary to keep captive fish in goodhealth. Fish food may be in the form of a flake, pellet or tablet.Pelleted forms, some of which sink rapidly, are often used for largerfish or bottom feeding species. Some fish foods also contain additives,such as beta carotene or sex hormones, to artificially enhance the colorof ornamental fish.

The term feed in the present invention also encompasses in someembodiment bird food. Bird food includes food that is used both inbirdfeeders and to feed pet birds. Typically bird food comprises of avariety of seeds, but may also encompass suet (beef or mutton fat).

As used herein the term “contacted” refers to the indirect or directapplication of the composition of the present invention to the product(e.g. the feed). Examples of the application methods which may be used,include, but are not limited to, treating the product in a materialcomprising the feed additive composition, direct application by mixingthe feed additive composition with the product, spraying the feedadditive composition onto the product surface or dipping the productinto a preparation of the feed additive composition.

In one embodiment the feed additive composition of the present inventionis preferably admixed with the product (e.g. feedstuff). Alternatively,the feed additive composition may be included in the emulsion or rawingredients of a feedstuff.

For some applications, it is important that the composition is madeavailable on or to the surface of a product to be affected/treated. Thisallows the composition to impart one or more of the following favourablecharacteristics: performance benefits.

The feed additive compositions of the present invention may be appliedto intersperse, coat and/or impregnate a product (e.g. feedstuff or rawingredients of a feedstuff) with a controlled amount of DFM and enzymes.

The DFM and enzymes may be used simultaneously (e.g. when they are inadmixture together or even when they are delivered by different routes)or sequentially (e.g. they may be delivered by different routes). In oneembodiment preferably the DFM and enzymes are applied simultaneously.Preferably the DFM and enzymes are admixed prior to being delivered to afeedstuff or to a raw ingredient of a feedstuff.

The DFM in feed additive compositions according to the presentinvention—can be added in suitable concentrations—such as for example inconcentrations in the final feed product which offer a daily dose ofbetween about 2×10⁵ CFU to about 2×10¹¹ CFU, suitably between about2×10⁶ to about 1×10¹⁰, suitably between about 3.75×10⁷ CFU to about1×10¹⁰ CFU.

Preferably, the feed additive composition of the present invention willbe thermally stable to heat treatment up to about 70° C.; up to about85° C.; or up to about 95° C. The heat treatment may be performed for upto about 1 minute; up to about 5 minutes; up to about 10 minutes; up toabout 30 minutes; up to about 60 minutes. The term thermally stablemeans that at least about 75% of the enzyme components and/or DFM thatwere present/active in the additive before heating to the specifiedtemperature are still present/active after it cools to room temperature.Preferably, at least about 80% of the enzyme components and/or DFM thatwere present and active in the additive before heating to the specifiedtemperature are still present and active after it cools to roomtemperature.

In a particularly preferred embodiment the feed additive composition ishomogenized to produce a powder.

In an alternative preferred embodiment, the feed additive composition isformulated to granules as described in WO2007/044968 (referred to as TPTgranules) incorporated herein by reference.

In another preferred embodiment when the feed additive composition isformulated into granules the granules comprise a hydrated barrier saltcoated over the protein core. The advantage of such salt coating isimproved thermo-tolerance, improved storage stability and protectionagainst other feed additives otherwise having adverse effect on theenzyme and/or DFM.

Preferably, the salt used for the salt coating has a water activitygreater than 0.25 or constant humidity greater than 60% at 20° C.

Preferably, the salt coating comprises a Na₂SO₄.

The method of preparing a feed additive composition may also comprisethe further step of pelleting the powder. The powder may be mixed withother components known in the art. The powder, or mixture comprising thepowder, may be forced through a die and the resulting strands are cutinto suitable pellets of variable length.

Optionally, the pelleting step may include a steam treatment, orconditioning stage, prior to formation of the pellets. The mixturecomprising the powder may be placed in a conditioner, e.g. a mixer withsteam injection. The mixture is heated in the conditioner up to aspecified temperature, such as from 60-100° C., typical temperatureswould be 70° C., 80° C., 85° C., 90° C. or 95° C. The residence time canbe variable from seconds to minutes and even hours. Such as 5 seconds,10 seconds, 15 seconds, 30 seconds, 1 minutes 2 minutes, 5 minutes, 10minutes, 15 minutes, 30 minutes and 1 hour.

It will be understood that the feed additive composition of the presentinvention is suitable for addition to any appropriate feed material.

As used herein, the term feed material refers to the basic feed materialto be consumed by an animal. It will be further understood that this maycomprise, for example, at least one or more unprocessed grains, and/orprocessed plant and/or animal material such as soybean meal or bonemeal.

As used herein, the term “feedstuff” refers to a feed material to whichone or more feed additive compositions have been added.

It will be understood by the skilled person that different animalsrequire different feedstuffs, and even the same animal may requiredifferent feedstuffs, depending upon the purpose for which the animal isreared.

Preferably, the feedstuff may comprise feed materials comprising maizeor corn, wheat, barley, triticale, rye, rice, tapioca, sorghum, and/orany of the by-products, as well as protein rich components like soybeanmean, rape seed meal, canola meal, cotton seed meal, sunflower seedmean, animal-by-product meals and mixtures thereof. More preferably, thefeedstuff may comprise animal fats and/or vegetable oils.

Optionally, the feedstuff may also contain additional minerals such as,for example, calcium and/or additional vitamins.

Preferably, the feedstuff is a corn soybean meal mix.

In one embodiment, preferably the feed is not pet food.

In another aspect there is provided a method for producing a feedstuff.Feedstuff is typically produced in feed mills in which raw materials arefirst ground to a suitable particle size and then mixed with appropriateadditives. The feedstuff may then be produced as a mash or pellets; thelater typically involves a method by which the temperature is raised toa target level and then the feed is passed through a die to producepellets of a particular size. The pellets are allowed to cool.Subsequently liquid additives such as fat and enzyme may be added.Production of feedstuff may also involve an additional step thatincludes extrusion or expansion prior to pelleting—in particular bysuitable techniques that may include at least the use of steam.

The feedstuff may be a feedstuff for a monogastric animal, such aspoultry (for example, broiler, layer, broiler breeders, turkey, duck,geese, water fowl), swine (all age categories), a pet (for example dogs,cats) or fish, preferably the feedstuff is for poultry.

In one embodiment the feedstuff is not for a layer.

By way of example only a feedstuff for chickens, e.g. broiler chickensmay be comprises of one or more of the ingredients listed in the tablebelow, for example in the % ages given in the table below:

Ingredients Starter (%) Finisher (%) Maize 46.2 46.7 Wheat Middlings 6.710.0 Maize DDGS 7.0 7.0 Soyabean Meal 48% CP 32.8 26.2 An/Veg Fat blend3.0 5.8 L-Lysine HCl 0.3 0.3 DL-methionine 0.3 0.3 L-threonine 0.1 0.1Salt 0.3 0.4 Limestone 1.1 1.1 Dicalcium Phosphate 1.2 1.2 PoultryVitamins and Micro- 0.3 0.3 minerals

By way of example only the diet specification for chickens, such asbroiler chickens, may be as set out in the Table below:

Diet specification Crude Protein (%) 23.00 20.40 Metabolizable EnergyPoultry 2950 3100 (kcal/kg) Calcium (%) 0.85 0.85 Available Phosphorus(%) 0.38 0.38 Sodium (%) 0.18 0.19 Dig. Lysine (%) 1.21 1.07 Dig.Methionine (%) 0.62 0.57 Dig. Methionine + Cysteine (%) 0.86 0.78 Dig.Threonine (%) 0.76 0.68

By way of example only a feedstuff laying hens may be comprises of oneor more of the ingredients listed in the table below, for example in the% ages given in the table below:

Ingredient Laying phase (%) Maize 10.0 Wheat 53.6 Maize DDGS 5.0 SoybeanMeal 48% CP 14.9 Wheat Middlings 3.0 Soybean Oil 1.8 L-Lysine HCl 0.2DL-methionine 0.2 L-threonine 0.1 Salt 0.3 Dicalcium Phosphate 1.6Limestone 8.9 Poultry Vitamins and Micro- 0.6 minerals

By way of example only the diet specification for laying hens may be asset out in the Table below:

Diet specification Crude Protein (%) 16.10 Metabolizable Energy Poultry2700 (kcal/kg) Lysine (%) 0.85 Methionine (%) 0.42 Methionine + Cysteine(%) 0.71 Threonine (%) 0.60 Calcium (%) 3.85 Available Phosphorus (%)0.42 Sodium (%) 0.16

By way of example only a feedstuff for turkeys may be comprises of oneor more of the ingredients listed in the table below, for example in the% ages given in the table below:

Phase 1 Phase 2 Phase 3 Phase 4 Ingredient (%) (%) (%) (%) Wheat 33.642.3 52.4 61.6 Maize DDGS 7.0 7.0 7.0 7.0 Soyabean Meal 48% CP 44.6 36.627.2 19.2 Rapeseed Meal 4.0 4.0 4.0 4.0 Soyabean Oil 4.4 4.2 3.9 3.6L-Lysine HCl 0.5 0.5 0.4 0.4 DL-methionine 0.4 0.4 0.3 0.2 L-threonine0.2 0.2 0.1 0.1 Salt 0.3 0.3 0.3 0.3 Limestone 1.0 1.1 1.1 1.0 DicalciumPhosphate 3.5 3.0 2.7 2.0 Poultry Vitamins and 0.4 0.4 0.4 0.4Micro-minerals

By way of example only the diet specification for turkeys may be as setout in the Table below:

Diet specification Crude Protein (%) 29.35 26.37 22.93 20.00Metabolizable Energy Poultry 2.850 2.900 2.950 3.001 (kcal/kg) Calcium(%) 1.43 1.33 1.22 1.02 Available Phosphorus (%) 0.80 0.71 0.65 0.53Sodium (%) 0.16 0.17 0.17 0.17 Dig. Lysine (%) 1.77 1.53 1.27 1.04 Dig.Methionine (%) 0.79 0.71 0.62 0.48 Dig. Methionine + Cysteine (%) 1.121.02 0.90 0.74 Dig. Threonine (%) 1.03 0.89 0.73 0.59

By way of example only a feedstuff for piglets may be comprises of oneor more of the ingredients listed in the table below, for example in the% ages given in the table below:

Ingredient Phase 1 (%) Phase 2 (%) Maize 20.0 7.0 Wheat 25.9 46.6 Rye4.0 10.0 Wheat middlings 4.0 4.0 Maize DDGS 6.0 8.0 Soyabean Meal 48% CP25.7 19.9 Dried Whey 10.0 0.0 Soyabean Oil 1.0 0.7 L-Lysine HCl 0.4 0.5DL-methionine 0.2 0.2 L-threonine 0.1 0.2 L-tryptophan 0.03 0.04Limestone 0.6 0.7 Dicalcium Phosphate 1.6 1.6 Swine Vitamins and Micro-0.2 0.2 minerals Salt 0.2 0.4

By way of example only the diet specification for piglets may be as setout in the Table below:

Diet specification Crude Protein (%) 21.50 20.00 Swine Digestible Energy3380 3320 (kcal/kg) Swine Net Energy (kcal/kg) 2270 2230 Calcium (%)0.80 0.75 Digestible Phosphorus (%) 0.40 0.35 Sodium (%) 0.20 0.20 Dig.Lysine (%) 1.23 1.14 Dig. Methionine (%) 0.49 0.44 Dig. Methionine +Cysteine (%) 0.74 0.68 Dig. Threonine (%) 0.80 0.74

By way of example only a feedstuff for grower/finisher pigs may becomprises of one or more of the ingredients listed in the table below,for example in the % ages given in the table below:

Ingredient Grower/Finisher (%) Maize 27.5 Soyabean Meal 48% CP 15.4Maize DDGS 20.0 Wheat bran 11.1 Rice bran 12.0 Canola seed meal 10.0Limestone 1.6 Dicalcium phosphate 0.01 Salt 0.4 Swine Vitamins andMicro-minerals 0.3 Lysine-HCl 0.2 Vegetable oil 0.5

By way of example only the diet specification for grower/finisher pigsmay be as set out in the Table below:

Diet specification Crude Protein (%) 22.60 Swine Metabolizable Energy3030 (kcal/kg) Calcium (%) 0.75 Available Phosphorus (%) 0.29 DigestibleLysine (%) 1.01 Dig. Methionine + Cysteine (%) 0.73 Digestible Threonine(%) 0.66

Forms

The feed additive composition of the present invention and othercomponents and/or the feedstuff comprising same may be used in anysuitable form.

The feed additive composition of the present invention may be used inthe form of solid or liquid preparations or alternatives thereof.Examples of solid preparations include powders, pastes, boluses,capsules, pellets, tablets, dusts, and granules which may be wettable,spray-dried or freeze-dried. Examples of liquid preparations include,but are not limited to, aqueous, organic or aqueous-organic solutions,suspensions and emulsions.

In some applications, DFM or feed additive compositions of the presentinvention may be mixed with feed or administered in the drinking water.In one embodiment the dosage range for inclusion into water is about1×10³ CFU/animal/day to about 1×10¹⁰ CFU/animal/day, and more preferablyabout 1×10⁷ CFU/animal/day.

Suitable examples of forms include one or more of: powders, pastes,boluses, pellets, tablets, pills, capsules, ovules, solutions orsuspensions, which may contain flavouring or colouring agents, forimmediate-, delayed-, modified-, sustained-, pulsed- orcontrolled-release applications.

By way of example, if the composition of the present invention is usedin a solid, e.g. pelleted form, it may also contain one or more of:excipients such as microcrystalline cellulose, lactose, sodium citrate,calcium carbonate, dibasic calcium phosphate and glycine; disintegrantssuch as starch (preferably corn, potato or tapioca starch), sodiumstarch glycollate, croscarmellose sodium and certain complex silicates;granulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),sucrose, gelatin and acacia; lubricating agents such as magnesiumstearate, stearic acid, glyceryl behenate and talc may be included.

Examples of nutritionally acceptable carriers for use in preparing theforms include, for example, water, salt solutions, alcohol, silicone,waxes, petroleum jelly, vegetable oils, polyethylene glycols, propyleneglycol, liposomes, sugars, gelatin, lactose, amylose, magnesiumstearate, talc, surfactants, silicic acid, viscous paraffin, perfumeoil, fatty acid monoglycerides and diglycerides, petroethral fatty acidesters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

Preferred excipients for the forms include lactose, starch, a cellulose,milk sugar or high molecular weight polyethylene glycols.

For aqueous suspensions and/or elixirs, the composition of the presentinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, propylene glycol and glycerin, andcombinations thereof.

Non-hydroscopic whey is often used as a carrier for DFMs (particularlybacterial DFMs) and is a good medium to initiate growth.

Bacterial DFM containing pastes may be formulated with vegetable oil andinert gelling ingredients.

Fungal products may be formulated with grain by-products as carriers.

In one embodiment preferably the feed additive composition according tothe present invention is not in the form of a microparticle system, suchas the microparticle system taught in WO2005/123034.

Dosing

The DFM and/or feed additive composition according to the presentinvention may be designed for one-time dosing or may be designed forfeeding on a daily basis.

The optimum amount of the composition (and each component therein) to beused in the combination of the present invention will depend on theproduct to be treated and/or the method of contacting the product withthe composition and/or the intended use for the same.

The amount of DFM and enzymes used in the compositions should be asufficient amount to be effective and to remain sufficiently effectivein improving the performance of the animal fed feed products containingsaid composition. This length of time for effectiveness should extend upto at least the time of utilisation of the product (e.g. feed additivecomposition or feed containing same).

The ratio of DFM to each enzyme in the feed can be in the ranges givenbelow:

DFM:phytase (CFU/FTU): In range from 5.0×10² CFU DFM:1 FTU enzyme to5.0×10⁹ CFU:1 FTU enzyme; preferably in the range from 7.5×10⁴ CFU DFM:1FTU enzyme to 2.5×10⁷ CFU:1 FTU enzyme.DFM: xylanase (CFU/XU): In range from 6.25×10¹ CFU DFM:1 XU enzyme to2.0×10⁹ CFU:1 XU enzyme; preferably in the range from 1.88×10⁴ CFU DFM:1XU enzyme to 1.0×10⁷ CFU:1 XU enzyme.DFM:amylase (CFU/AU): In range from 1.0×10² CFU DFM:1 AU enzyme to2.0×10¹⁰ CFU:1 AU enzyme; preferably in the range from 3.7×10⁴ CFU DFM:1AU enzyme to 1.0×10⁸ CFU:1 AU enzyme.DFM:protease (CFU/PU): In range from 5.0×10¹ CFU DFM:1 PU enzyme to1.0×10⁹ CFU:1 PU enzyme; preferably in the range from 1.25×10⁴ CFU DFM:1PU enzyme to 5.0×10⁶ CFU:1 PU enzyme.

In one embodiment preferably the feedstuff comprises the following:

a protease at at least 4000 PU/kg of feed;a xylanase at at least 1000 XU/kg to 2000 XU/kg of feed (e.g. Avizyme at1000 XU/kg of feed or Axtra XAP at at least 2000 XU/kg of feed);an amylase; at least 1800 AU/kg or 200 TAU/kg of feed (e.g. Avizyme at1800 AU/kg or Axtra XAP at at least 200 TAU/kg of feed);a phytase at at least 500 FTU/kg of feed; andEnvivo Pro (DFM) at at least 75,000 CFU/g to 150,000 CFU/g of feed.

In one embodiment preferably the feedstuff comprises the following:

a protease at 4000 PU/kg of feed;a xylanase at 1000 XU/kg to 2000 XU/kg of feed (e.g. Avizyme at 1000XU/kg of feed or Axtra XAP at 2000 XU/kg of feed);an amylase; 1800 AU/kg or 200 TAU/kg of feed (e.g. Avizyme at 1800 AU/kgor Axtra XAP at 200 TAU/kg of feed);a phytase at 500 FTU/kg of feed; andEnvivo Pro (DFM) at 75,000 CFU/g to 150,000 CFU/g of feed.

In one embodiment preferably the feedstuff comprises the following:

a protease at 5000 PU/kg of feed;a xylanase at 1250 XU/kg to 2500 XU/kg of feed (e.g. Avizyme at 1000XU/kg of feed or Axtra XAP at 2500 XU/kg of feed);an amylase; 2250 AU/kg or 250 TAU/kg of feed (e.g. Avizyme at 1800 AU/kgor Axtra XAP at 250 TAU/kg of feed);a phytase at 625 FTU/kg of feed; andEnvivo Pro (DFM) at 75,000 CFU/g to 150,000 CFU/g of feed.

In another embodiment the feedstuff comprises the following:

a protease at 2000 PU/kg of feed;a xylanase at 500 XU/kg to 1000 XU/kg of feed (e.g. Avizyme at 500 XU/kgof feed or Axtra XAP at 1000 XU/kg of feed);an amylase; 900 AU/kg or 100 TAU/kg of feed (e.g. Avizyme at 900 AU/kgor Axtra XAP at 100 TAU/kg of feed);a phytase at 500 FTU/kg of feed; andEnvivo Pro (DFM) at 37,500 CFU/g to 75,000 CFU/g of feed.

In a preferred embodiment the feed additive composition comprisessufficient enzyme and DFMs to dose the feedstuff as follows:

a protease at 4000 PU/kg of feed;a xylanase at 1000 XU/kg to 2000 XU/kg of feed (e.g. Avizyme at 1000XU/kg of feed or Axtra XAP at 2000 XU/kg of feed);an amylase; 1800 AU/kg or 200 TAU/kg of feed (e.g. Avizyme at 1800 AU/kgor Axtra XAP at 200 TAU/kg of feed);a phytase at 500 FTU/kg of feed; andEnvivo Pro (DFM) at 75,000 CFU/g to 150,000 CFU/g of feed.

In a preferred embodiment the feed additive composition comprisessufficient enzyme and DFMs to dose the feedstuff as follows:

a protease at 2000 PU/kg of feed;a xylanase at 500 XU/kg to 1000 XU/kg of feed (e.g. Avizyme at 500 XU/kgof feed or Axtra XAP at 1000 XU/kg of feed);an amylase; 900 AU/kg or 100 TAU/kg of feed (e.g. Avizyme at 900 AU/kgor Axtra XAP at 100 TAU/kg of feed);a phytase at 500 FTU/kg of feed; andEnvivo Pro (DFM) at 37,500 CFU/g to 75,000 CFU/g of feed.Combination with Other Components

The DFM and enzyme(s) for use in the present invention may be used incombination with other components. Thus, the present invention alsorelates to combinations. The DFM in combination with a protease,xylanase, amylase and phytase may be referred to herein as “the feedadditive composition of the present invention”.

The combination of the present invention comprises the feed additivecomposition of the present invention (or one or more of the constituentsthereof) and another component which is suitable for animal consumptionand is capable of providing a medical or physiological benefit to theconsumer.

In one embodiment preferably the “another component” is not a furtherenzyme or a further DFM.

The components may be prebiotics. Prebiotics are typicallynon-digestible carbohydrate (oligo- or polysaccharides) or a sugaralcohol which is not degraded or absorbed in the upper digestive tract.Known prebiotics used in commercial products and useful in accordancewith the present invention include inulin (fructo-oligosaccharide, orFOS) and transgalacto-oligosaccharides (GOS or TOS). Suitable prebioticsinclude palatinoseoligosaccharide, soybean oligosaccharide, alginate,xanthan, pectin, locust bean gum (LBG), inulin, guar gum,galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS),non-degradable starch, lactosaccharose, lactulose, lactitol, maltitol,maltodextrin, polydextrose (i.e. Litesse®), lactitol, lactosucrose,soybean oligosaccharides, palatinose, isomalto-oligosaccharides,gluco-oligosaccharides and xylo-oligosaccharides, pectin fragments,dietary fibres, mannan-oligosaccharides.

Dietary fibres may include non-starch polysaccharides, such asarabinoxylans, cellulose and many other plant components, such asresistant dextrins, inulin, lignin, waxes, chitins, pectins,beta-glucans and oligosaccharides.

In one embodiment the present invention relates to the combination ofthe feed additive composition (or one or more of the constituentsthereof) according to the present invention with a prebiotic. In anotherembodiment the present invention relates to a feed additive compositioncomprising (or consisting essentially of or consisting of) a DFM incombination with a xylanase, an amylase, a phytase, a protease and aprebiotic.

The prebiotic may be administered simultaneously with (e.g. in admixturetogether with or delivered simultaneously by the same or differentroutes) or sequentially to (e.g. by the same or different routes) thefeed additive composition (or constituents thereof) according to thepresent invention.

Other components of the combinations of the present invention includepolydextrose, such as Litesse®, and/or a maltodextrin and/or lactitol.These other components may be optionally added to the feed additivecomposition to assist the drying process and help the survival of DFM.

Further examples of other suitable components include one or more of:thickeners, gelling agents, emulsifiers, binders, crystal modifiers,sweeteners (including artificial sweeteners), rheology modifiers,stabilisers, anti-oxidants, dyes, enzymes, carriers, vehicles,excipients, diluents, lubricating agents, flavouring agents, colouringmatter, suspending agents, disintegrants, granulation binders etc. Theseother components may be natural. These other components may be preparedby use of chemical and/or enzymatic techniques.

In one embodiment the DFM and/or enzymes may be encapsulated. In oneembodiment the feed additive composition and/or DFM and/or enzymesis/are formulated as a dry powder or granule as described inWO2007/044968 (referred to as TPT granules)—reference incorporatedherein by reference.

In one preferred embodiment the DFM and/or enzymes for use in thepresent invention may be used in combination with one or more lipids.

For example, the DFM and/or enzymes for use in the present invention maybe used in combination with one or more lipid micelles. The lipidmicelle may be a simple lipid micelle or a complex lipid micelle.

The lipid micelle may be an aggregate of orientated molecules ofamphipathic substances, such as a lipid and/or an oil.

As used herein the term “thickener or gelling agent” refers to a productthat prevents separation by slowing or preventing the movement ofparticles, either droplets of immiscible liquids, air or insolublesolids. Thickening occurs when individual hydrated molecules cause anincrease in viscosity, slowing the separation. Gelation occurs when thehydrated molecules link to form a three-dimensional network that trapsthe particles, thereby immobilising them.

The term “stabiliser” as used here is defined as an ingredient orcombination of ingredients that keeps a product (e.g. a feed product)from changing over time.

The term “emulsifier” as used herein refers to an ingredient (e.g. afeed ingredient) that prevents the separation of emulsions. Emulsionsare two immiscible substances, one present in droplet form, containedwithin the other. Emulsions can consist of oil-in-water, where thedroplet or dispersed phase is oil and the continuous phase is water; orwater-in-oil, where the water becomes the dispersed phase and thecontinuous phase is oil. Foams, which are gas-in-liquid, andsuspensions, which are solid-in-liquid, can also be stabilised throughthe use of emulsifiers.

As used herein the term “binder” refers to an ingredient (e.g. a feedingredient) that binds the product together through a physical orchemical reaction. During “gelation” for instance, water is absorbed,providing a binding effect. However, binders can absorb other liquids,such as oils, holding them within the product. In the context of thepresent invention binders would typically be used in solid orlow-moisture products for instance baking products: pastries, doughnuts,bread and others.

“Carriers” or “vehicles” mean materials suitable for administration ofthe DFM and/or enzymes and include any such material known in the artsuch as, for example, any liquid, gel, solvent, liquid diluent,solubilizer, or the like, which is non-toxic and which does not interactwith any components of the composition in a deleterious manner.

The present invention provides a method for preparing a feed additivecomposition comprising admixing a DFM, a xylanase, a protease, a phytaseand a amylase with at least one physiologically acceptable carrierselected from at least one of maltodextrin, limestone (calciumcarbonate), cyclodextrin, wheat or a wheat component, sucrose, starch,Na₂SO₄, Talc, PVA, sorbitol, benzoate, sorbiate, glycerol, sucrose,propylene glycol, 1,3-propane diol, glucose, parabens, sodium chloride,citrate, acetate, phosphate, calcium, metabisulfite, formate andmixtures thereof.

Examples of excipients include one or more of: microcrystallinecellulose and other celluloses, lactose, sodium citrate, calciumcarbonate, dibasic calcium phosphate, glycine, starch, milk sugar andhigh molecular weight polyethylene glycols.

Examples of disintegrants include one or more of: starch (preferablycorn, potato or tapioca starch), sodium starch glycollate,croscarmellose sodium and certain complex silicates.

Examples of granulation binders include one or more of:polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia.

Examples of lubricating agents include one or more of: magnesiumstearate, stearic acid, glyceryl behenate and talc.

Examples of diluents include one or more of: water, ethanol, propyleneglycol and glycerin, and combinations thereof.

The other components may be used simultaneously (e.g. when they are inadmixture together or even when they are delivered by different routes)or sequentially (e.g. they may be delivered by different routes).

Preferably, when the feed additive composition of the present inventionis admixed with another component(s), the DFM remains viable.

In one embodiment preferably the feed additive composition according tothe present invention does not comprise chromium or organic chromium

In one embodiment preferably the feed additive according to the presentinvention does not contain glucanase.

In one embodiment preferably the feed additive according to the presentinvention does not contain sorbic acid.

Concentrates

The DFMs for use in the present invention may be in the form ofconcentrates. Typically these concentrates comprise a substantially highconcentration of a DFM.

Feed additive compositions according to the present invention may have acontent of viable cells (colony forming units, CFUs) which is in therange of at least 10⁴ CFU/g (suitably including at least 10⁵ CFU/g, suchas at least 10⁶ CFU/g, e.g. at least 10⁷ CFU/g, at least 10⁸ CFU/g, e.g.at least 10⁹ CFU/g) to about 10¹⁰ CFU/g (or even about 10¹¹ CFU/g orabout 10¹² CFU/g).

When the DFM is in the form of a concentrate the feed additivecompositions according to the present invention may have a content ofviable cells in the range of at least 10⁹ CFU/g to about 10¹² CFU/g,preferably at least 10¹⁰ CFU/g to about 10¹² CFU/g.

Powders, granules and liquid compositions in the form of concentratesmay be diluted with water or resuspended in water or other suitablediluents, for example, an appropriate growth medium such as milk ormineral or vegetable oils, to give compositions ready for use.

The DFM or feed additive composition of the present invention or thecombinations of the present invention in the form of concentrates may beprepared according to methods known in the art.

In one aspect of the present invention the enzymes or feed is contactedby a composition in a concentrated form.

The compositions of the present invention may be spray-dried orfreeze-dried by methods known in the art.

Typical processes for making particles using a spray drying processinvolve a solid material which is dissolved in an appropriate solvent(e.g. a culture of a DFM in a fermentation medium). Alternatively, thematerial can be suspended or emulsified in a non-solvent to form asuspension or emulsion. Other ingredients (as discussed above) orcomponents such as anti-microbial agents, stabilising agents, dyes andagents assisting with the drying process may optionally be added at thisstage.

The solution then is atomised to form a fine mist of droplets. Thedroplets immediately enter a drying chamber where they contact a dryinggas. The solvent is evaporated from the droplets into the drying gas tosolidify the droplets, thereby forming particles. The particles are thenseparated from the drying gas and collected.

Subject

The term “subject”, as used herein, means an animal that is to be or hasbeen administered with a feed additive composition according to thepresent invention or a feedstuff comprising said feed additivecomposition according to the present invention.

The term “subject”, as used herein, means an animal. Preferably, thesubject is a mammal, bird, fish or crustacean including for examplelivestock or a domesticated animal (e.g. a pet).

In one embodiment the “subject” is livestock.

The term “livestock”, as used herein refers to any farmed animal.Preferably, livestock is one or more of cows or bulls (includingcalves), poultry, pigs (including piglets), poultry (including broilers,chickens and turkeys), birds, fish (including freshwater fish, such assalmon, cod, trout and carp, e.g. koi carp, and marine fish, such as seabass), crustaceans (such as shrimps, mussels and scallops), horses(including race horses), sheep (including lambs).

In one embodiment the term livestock and/or poultry and/or chickens doesnot include egg layers.

In another embodiment the “subject” is a domesticated animal or pet oran animal maintained in a zoological environment.

The term “domesticated animal or pet or animal maintained in azoological environment” as used herein refers to any relevant animalincluding canines (e.g. dogs), felines (e.g. cats), rodents (e.g. guineapigs, rats, mice), birds, fish (including freshwater fish and marinefish), and horses.

In one embodiment the subject may be challenged by an enteric pathogen.

By way of example a subject may have one or more enteric pathogenspresent in its gut or digestive tract. For example a subject may haveone or more enteric pathogens in its gut or digestive tract at a levelwhich:

-   -   i) results in loss of performance of the animal and/or    -   ii) is at clinically relevant levels; or    -   iii) is at sub-clinical levels.

The enteric pathogen may be Clostridium perfringens for example.

Performance

As used herein, “animal performance” may be determined by the feedefficiency and/or weight gain of the animal and/or by the feedconversion ratio and/or by the digestibility of a nutrient in a feed(e.g. amino acid digestibility) and/or digestible energy ormetabolizable energy in a feed and/or by nitrogen retention and/or byanimals ability to avoid the negative effects of necrotic enteritisand/or by the immune response of the subject.

Preferably “animal performance” is determined by feed efficiency and/orweight gain of the animal and/or by the feed conversion ratio.

By “improved animal performance” it is meant that there is increasedfeed efficiency, and/or increased weight gain and/or reduced feedconversion ratio and/or improved digestibility of nutrients or energy ina feed and/or by improved nitrogen retention and/or by improved abilityto avoid the negative effects of necrotic enteritis and/or by animproved immune response in the subject resulting from the use of feedadditive composition of the present invention in feed in comparison tofeed which does not comprise said feed additive composition.

Preferably, by “improved animal performance” it is meant that there isincreased feed efficiency and/or increased weight gain and/or reducedfeed conversion ratio.

As used herein, the term “feed efficiency” refers to the amount ofweight gain in an animal that occurs when the animal is fed ad-libitumor a specified amount of food during a period of time.

By “increased feed efficiency” it is meant that the use of a feedadditive composition according the present invention in feed results inan increased weight gain per unit of feed intake compared with an animalfed without said feed additive composition being present.

Feed Conversion Ratio (FCR)

As used herein, the term “feed conversion ratio” refers to the amount offeed fed to an animal to increase the weight of the animal by aspecified amount.

An improved feed conversion ratio means a lower feed conversion ratio.

By “lower feed conversion ratio” or “improved feed conversion ratio” itis meant that the use of a feed additive composition in feed results ina lower amount of feed being required to be fed to an animal to increasethe weight of the animal by a specified amount compared to the amount offeed required to increase the weight of the animal by the same amountwhen the feed does not comprise said feed additive composition.

Nutrient Digestibility

Nutrient digestibility as used herein means the fraction of a nutrientthat disappears from the gastro-intestinal tract or a specified segmentof the gastro-intestinal tract, e.g. the small intestine. Nutrientdigestibility may be measured as the difference between what isadministered to the subject and what comes out in the faeces of thesubject, or between what is administered to the subject and what remainsin the digesta on a specified segment of the gastro intestinal tract,e.g. the ileum.

Nutrient digestibility as used herein may be measured by the differencebetween the intake of a nutrient and the excreted nutrient by means ofthe total collection of excreta during a period of time; or with the useof an inert marker that is not absorbed by the animal, and allows theresearcher calculating the amount of nutrient that disappeared in theentire gastro-intestinal tract or a segment of the gastro-intestinaltract. Such an inert marker may be titanium dioxide, chromic oxide oracid insoluble ash. Digestibility may be expressed as a percentage ofthe nutrient in the feed, or as mass units of digestible nutrient permass units of nutrient in the feed.

Nutrient digestibility as used herein encompasses starch digestibility,fat digestibility, protein digestibility, and amino acid digestibility.

Energy digestibility as used herein means the gross energy of the feedconsumed minus the gross energy of the faeces or the gross energy of thefeed consumed minus the gross energy of the remaining digesta on aspecified segment of the gastro-intestinal tract of the animal, e.g. theileum. Metabolizable energy as used herein refers to apparentmetabolizable energy and means the gross energy of the feed consumedminus the gross energy contained in the faeces, urine, and gaseousproducts of digestion. Energy digestibility and metabolizable energy maybe measured as the difference between the intake of gross energy and thegross energy excreted in the faeces or the digesta present in specifiedsegment of the gastro-intestinal tract using the same methods to measurethe digestibility of nutrients, with appropriate corrections fornitrogen excretion to calculate metabolizable energy of feed.

Nitrogen Retention

Nitrogen retention as used herein means as subject's ability to retainnitrogen from the diet as body mass. A negative nitrogen balance occurswhen the excretion of nitrogen exceeds the daily intake and is oftenseen when the muscle is being lost. A positive nitrogen balance is oftenassociated with muscle growth, particularly in growing animals.

Nitrogen retention may be measured as the difference between the intakeof nitrogen and the excreted nitrogen by means of the total collectionof excreta and urine during a period of time. It is understood thatexcreted nitrogen includes undigested protein from the feed, endogenousproteinaceous secretions, microbial protein, and urinary nitrogen.

Survival

The term survival as used herein means the number of subject remainingalive. The term “improved survival” may be another way of saying“reduced mortality”.

Carcass Yield and Meat Yield

The term carcass yield as used herein means the amount of carcass as aproportion of the live body weight, after a commercial or experimentalprocess of slaughter. The term carcass means the body of an animal thathas been slaughtered for food, with the head, entrails, part of thelimbs, and feathers or skin removed. The term meat yield as used hereinmeans the amount of edible meat as a proportion of the live body weight,or the amount of a specified meat cut as a proportion of the live bodyweight.

Weight Gain

The present invention further provides a method of increasing weightgain in a subject, e.g. poultry or swine, comprising feeding saidsubject a feedstuff comprising a feed additive composition according tothe present invention.

An “increased weight gain” refers to an animal having increased bodyweight on being fed feed comprising a feed additive composition comparedwith an animal being fed a feed without said feed additive compositionbeing present.

Necrotic Enteritis

Necrotic enteritis is an acute or chronic enterotoxemia seen inchickens, turkeys and ducks worldwide, caused by Clostridiumperfringens. Necrotic enteritis is often characterised by afibrino-necrotic enteritis, usually of the mid-small intestine.Mortality may be 5-50%, usually around 10%. Infection occurs byfaecal-oral transmission. Spores of the causative organism are highlyresistant. Predisposing factors include coccidiosis/coccidiasis, diet(high protein), in ducks possibly heavy strains, high viscosity diets(often associated with high rye and wheat inclusions in the diet),contaminated feed and/or water, other debilitating diseases.

The present invention relates to increasing the subject's resistance tonecrotic enteritis. In other words, the present invention relates toavoiding or reducing the negative effect of necrotic enteritis.

The term “resistance to” as used herein may encompasses the term“tolerance of”. Therefore in one embodiment the subject may not beresistant to necrotic enteritis but the subject may be able to toleratethe necrotic enteritis, i.e. without negative effects on performance ofthe subject.

In one embodiment the present invention relates to a feed additivecomposition according to the present invention for treating orpreventing necrotic enteritis in a subject. Typically the subject willbe one which has been or will be challenged with Clostridium perfringensand/or Eimeria species. Such challenge may come from the environment, orthe application of live microorganisms in the feed or drinking water,e.g. when live coccidia vaccines are used.

In another embodiment the present invention relates to a feed additivecomposition for preventing and/or treating coccidiosis and/or necroticenteritis in a subject.

The present invention yet further provides a method of preventing and/ortreating necrotic enteritis and/or coccidiosis wherein an effectiveamount of a feed additive composition according to the present inventionis administered to a subject.

Immune Response

Immune response as used herein means one of the multiple ways in whichDFMs modulate the immune system of animals, including increased antibodyproduction, up-regulation of cell mediated immunity, up-regulation ofpro-inflammatory cytokines, and augmented toll-like receptor signalling.It is understood that immuno-stimulation of the gastro intestinal tractby DFMs may be advantageous to protect the host against disease, andthat immuno-suppression of the gastro intestinal tract may beadvantageous to the host because less nutrients and energy are used tosupport the immune function.

Preferably the immune response is a cellular immune response.

Preferably immune response is measure by looking at immune markers.

Pathogenic Bacteria

The term pathogenic bacteria as used herein means for example toxigenicclostridia species, e.g. Clostridium perfringens and/or E. coli and/orSalmonella spp and/or Campylobacter spp. In one embodiment thepathogenic bacteria may be Avian pathogenic E. coli species.

The present invention may reduce populations of pathogenic bacteria inthe gastrointestinal tract of a subject.

Nutrient Excretion

In one embodiment the present invention relates to reducing nutrientexcretion in manure. This has positive effects on reducing environmentalhazards. For example, in a preferred embodiment the present inventionrelates to reducing nitrogen and/or phosphorus content in the subject'smanure. This, therefore, reduces the amount of nitrogen and/orphosphorus in the environment, which can be beneficial.

Probiotic

For some applications, it is believed that the DFM in the composition ofthe present invention can exert a probiotic culture effect. It is alsowithin the scope of the present invention to add to the composition ofthe present invention further probiotic and/or prebiotics.

Here, a prebiotic is:

“a non-digestible food ingredient that beneficially affects the host byselectively stimulating the growth and/or the activity of one or alimited number of beneficial bacteria”.

The term “probiotic culture” as used herein defines live microorganisms(including bacteria or yeasts for example) which, when for exampleingested or locally applied in sufficient numbers, beneficially affectsthe host organism, i.e. by conferring one or more demonstrable healthbenefits on the host organism. Probiotics may improve the microbialbalance in one or more mucosal surfaces. For example, the mucosalsurface may be the intestine, the urinary tract, the respiratory tractor the skin. The term “probiotic” as used herein also encompasses livemicroorganisms that can stimulate the beneficial branches of the immunesystem and at the same time decrease the inflammatory reactions in amucosal surface, for example the gut.

Whilst there are no lower or upper limits for probiotic intake, it hasbeen suggested that at least 10⁶-10¹², preferably at least 10⁶-10¹⁰,preferably 10⁸-10⁹, cfu as a daily dose will be effective to achieve thebeneficial health effects in a subject.

Isolated

In one aspect, suitably the enzyme or DFM used in the present inventionmay be in an isolated form. The term “isolated” means that the enzyme orDFM is at least substantially free from at least one other componentwith which the enzyme or DFM is naturally associated in nature and asfound in nature. The enzyme or DFM of the present invention may beprovided in a form that is substantially free of one or morecontaminants with which the substance might otherwise be associated.Thus, for example it may be substantially free of one or morepotentially contaminating polypeptides and/or nucleic acid molecules.

Purified

In one aspect, preferably the enzyme and/or DFM according to the presentinvention is in a purified form. The term “purified” means that theenzyme and/or DFM is present at a high level. The enzyme and/or DFM isdesirably the predominant component present in a composition.Preferably, it is present at a level of at least about 90%, or at leastabout 95% or at least about 98%, said level being determined on a dryweight/dry weight basis with respect to the total composition underconsideration.

It is envisaged within the scope of the present invention that theembodiments of the invention can be combined such that combinations ofany of the features described herein are included within the scope ofthe present invention. In particular, it is envisaged within the scopeof the present invention that any of the therapeutic effects of thebacteria may be exhibited concomitantly.

Nucleotide Sequence

The scope of the present invention encompasses nucleotide sequencesencoding proteins having the specific properties as defined herein.

The term “nucleotide sequence” as used herein refers to anoligonucleotide sequence or polynucleotide sequence, and variant,homologues, fragments and derivatives thereof (such as portionsthereof). The nucleotide sequence may be of genomic or synthetic orrecombinant origin, which may be double-stranded or single-strandedwhether representing the sense or anti-sense strand.

The term “nucleotide sequence” in relation to the present inventionincludes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it meansDNA, more preferably cDNA sequence coding for the present invention.

In a preferred embodiment, the nucleotide sequence when relating to andwhen encompassed by the per se scope of the present invention does notinclude the native nucleotide sequence according to the presentinvention when in its natural environment and when it is linked to itsnaturally associated sequence(s) that is/are also in its/their naturalenvironment. For ease of reference, we shall call this preferredembodiment the “non-native nucleotide sequence”. In this regard, theterm “native nucleotide sequence” means an entire nucleotide sequencethat is in its native environment and when operatively linked to anentire promoter with which it is naturally associated, which promoter isalso in its native environment. However, the amino acid sequenceencompassed by the scope of the present invention can be isolated and/orpurified post expression of a nucleotide sequence in its nativeorganism. Preferably, however, the amino acid sequence encompassed byscope of the present invention may be expressed by a nucleotide sequencein its native organism but wherein the nucleotide sequence is not underthe control of the promoter with which it is naturally associated withinthat organism.

Typically, the nucleotide sequence encompassed by the scope of thepresent invention is prepared using recombinant DNA techniques (i.e.recombinant DNA). However, in an alternative embodiment of theinvention, the nucleotide sequence could be synthesised, in whole or inpart, using chemical methods well known in the art (see Caruthers M H etal., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) NucAcids Res Symp Ser 225-232).

Preparation of the Nucleotide Sequence

A nucleotide sequence encoding either a protein which has the specificproperties as defined herein or a protein which is suitable formodification may be identified and/or isolated and/or purified from anycell or organism producing said protein. Various methods are well knownwithin the art for the identification and/or isolation and/orpurification of nucleotide sequences. By way of example, PCRamplification techniques to prepare more of a sequence may be used oncea suitable sequence has been identified and/or isolated and/or purified.

By way of further example, a genomic DNA and/or cDNA library may beconstructed using chromosomal DNA or messenger RNA from the organismproducing the enzyme. If the amino acid sequence of the enzyme is known,labelled oligonucleotide probes may be synthesised and used to identifyenzyme-encoding clones from the genomic library prepared from theorganism. Alternatively, a labelled oligonucleotide probe containingsequences homologous to another known enzyme gene could be used toidentify enzyme-encoding clones. In the latter case, hybridisation andwashing conditions of lower stringency are used. Alternatively,enzyme-encoding clones could be identified by inserting fragments ofgenomic DNA into an expression vector, such as a plasmid, transformingenzyme-negative bacteria with the resulting genomic DNA library, andthen plating the transformed bacteria onto agar plates containing asubstrate for enzyme (i.e. maltose), thereby allowing clones expressingthe enzyme to be identified.

In a yet further alternative, the nucleotide sequence encoding theenzyme may be prepared synthetically by established standard methods,e.g. the phosphoroamidite method described by Beucage S. L. et al.,(1981) Tetrahedron Letters 22, p 1859-1869, or the method described byMatthes et al., (1984) EMBO J. 3, p 801-805. In the phosphoroamiditemethod, oligonucleotides are synthesised, e.g. in an automatic DNAsynthesiser, purified, annealed, ligated and cloned in appropriatevectors.

The nucleotide sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin, or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate) in accordance with standard techniques. Each ligatedfragment corresponds to various parts of the entire nucleotide sequence.The DNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491).

Amino Acid Sequences

The scope of the present invention also encompasses amino acid sequencesof enzymes having the specific properties as defined herein.

As used herein, the term “amino acid sequence” is synonymous with theterm “polypeptide” and/or the term “protein”. In some instances, theterm “amino acid sequence” is synonymous with the term “peptide”. Insome instances, the term “amino acid sequence” is synonymous with theterm “enzyme”.

The amino acid sequence may be prepared/isolated from a suitable source,or it may be made synthetically or it may be prepared by use ofrecombinant DNA techniques.

The protein encompassed in the present invention may be used inconjunction with other proteins, particularly enzymes. Thus the presentinvention also covers a combination of proteins wherein the combinationcomprises the protein/enzyme of the present invention and anotherprotein/enzyme, which may be another protein/enzyme according to thepresent invention.

Preferably the amino acid sequence when relating to and when encompassedby the per se scope of the present invention is not a native enzyme. Inthis regard, the term “native enzyme” means an entire enzyme that is inits native environment and when it has been expressed by its nativenucleotide sequence.

Sequence Identity or Sequence Homology

The present invention also encompasses the use of sequences having adegree of sequence identity or sequence homology with amino acidsequence(s) of a polypeptide having the specific properties definedherein or of any nucleotide sequence encoding such a polypeptide(hereinafter referred to as a “homologous sequence(s)”). Here, the term“homologue” means an entity having a certain homology with the subjectamino acid sequences and the subject nucleotide sequences. Here, theterm “homology” can be equated with “identity”.

The homologous amino acid sequence and/or nucleotide sequence shouldprovide and/or encode a polypeptide which retains the functionalactivity and/or enhances the activity of the enzyme.

In the present context, a homologous sequence is taken to include anamino acid sequence which may be at least 75, 85 or 90% identical,preferably at least 95 or 98% identical to the subject sequence.Typically, the homologues will comprise the same active sites etc. asthe subject amino acid sequence. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity.

In the present context, a homologous sequence is taken to include anucleotide sequence which may be at least 75, 85 or 90% identical,preferably at least 95 or 98% identical to a nucleotide sequenceencoding a polypeptide of the present invention (the subject sequence).Typically, the homologues will comprise the same sequences that code forthe active sites etc. as the subject sequence. Although homology canalso be considered in terms of similarity (i.e. amino acid residueshaving similar chemical properties/functions), in the context of thepresent invention it is preferred to express homology in terms ofsequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the Vector NTI (Invitrogen Corp.). Examples of softwarethat can perform sequence comparisons include, but are not limited to,the BLAST package (see Ausubel et al 1999 Short Protocols in MolecularBiology, 4th Ed—Chapter 18), BLAST 2 (see FEMS Microbiol Lett 1999174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 andtatiana@ncbi.nlm.nih.gov), FASTA (Altschul et al 1990 J. Mol. Biol.403-410) and AlignX for example. At least BLAST, BLAST 2 and FASTA areavailable for offline and online searching (see Ausubel et al 1999,pages 7-58 to 7-60).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. Vector NTI programs generally use either the publicdefault values or a custom symbol comparison table if supplied (see usermanual for further details). For some applications, it is preferred touse the default values for the Vector NTI package.

Alternatively, percentage homologies may be calculated using themultiple alignment feature in Vector NTI (Invitrogen Corp.), based on analgorithm, analogous to CLUSTAL (Higgins DG & Sharp P M (1988), Gene73(1), 237-244).

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Should Gap Penalties be used when determining sequence identity, thenpreferably the following parameters are used for pairwise alignment:

FOR BLAST GAP OPEN 0 GAP EXTENSION 0 FOR CLUSTAL DNA PROTEIN WORD 2 1 Ktriple SIZE GAP 15 10 PENALTY GAP 6.66 0.1 EXTENSION

In one embodiment, CLUSTAL may be used with the gap penalty and gapextension set as defined above.

Suitably, the degree of identity with regard to a nucleotide sequence isdetermined over at least 20 contiguous nucleotides, preferably over atleast 30 contiguous nucleotides, preferably over at least 40 contiguousnucleotides, preferably over at least 50 contiguous nucleotides,preferably over at least 60 contiguous nucleotides, preferably over atleast 100 contiguous nucleotides.

Suitably, the degree of identity with regard to a nucleotide sequencemay be determined over the whole sequence.

Hybridisation

The present invention also encompasses sequences that are complementaryto the nucleic acid sequences of the present invention or sequences thatare capable of hybridising either to the sequences of the presentinvention or to sequences that are complementary thereto.

The term “hybridisation” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction (PCR) technologies.

The present invention also encompasses the use of nucleotide sequencesthat are capable of hybridising to the sequences that are complementaryto the sequences presented herein, or any derivative, fragment orderivative thereof.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hybridising to the nucleotide sequencespresented herein.

Preferably, complementary sequences are those capable of hybridisingunder stringent conditions (e.g. 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl,0.015 M Na₃citrate pH 7.0}) to the nucleotide sequences presentedherein.

More preferably, complementary sequences are those that are capable ofhybridising under high stringency conditions (e.g. 65° C. and 0.1×SSC{1×SSC=0.15 M NaCl, 0.015 M Na₃citrate pH 7.0}) to the nucleotidesequences presented herein.

In a more preferred aspect, the present invention covers nucleotidesequences that can hybridise to the nucleotide sequence of the presentinvention, or the complement thereof, under high stringent conditions(e.g. 65° C. and 0.1×SSC).

EXAMPLES Example 1 Materials and Methods

Three thousand six hundred one-day-old Cobb male chicks were purchasedfrom a commercial hatchery. At study initiation, fifty males wereallocated to each treatment pen by blocks. The study consisted of thefollowing treatments (Table 1):

TABLE 1 Experimental design of Example 1. Clostridium perfringensAdditional Treatment Challenge Phytase¹ enzyme² DFM³ 1 No 500 FTU/kgNone None 2 Yes 500 FTU/kg None None 3 Yes 500 FTU/kg Amylase None (200u/kg)  4 Yes 500 FTU/kg Protease None (5000 u/kg) 5 Yes 500 FTU/kgXylanase⁴ None (2000 u/kg) Amylase⁴ (200 u/kg)  Protease⁴ (5000 u/kg) 6Yes 500 FTU/kg None Enviva Pro (7.5 × 10⁴ CFU/g) 7 Yes 500 FTU/kgAmylase Enviva Pro (200 u/kg)  (7.5 × 10⁴ CFU/g) 8 Yes 500 FTU/kgProtease Enviva Pro (5000 u/kg) (7.5 × 10⁴ CFU/g) 9 Yes 500 FTU/kgXylanase⁴ Enviva Pro (2000 u/kg) (7.5 × 10⁴ CFU/g) Amylase⁴ (200 u/kg) Protease⁴ (5000 u/kg) ¹Phytase from E. coli. ²Amylase from Bacilluslicheniformis, xylanase from Trichoderma reesei, protease from Bacillussubtilis. ³Enviva Pro ® is combination of Bacillus subtilis strainsBs2084, LSSAO1 and 15AP4, provided by Danisco A/S. ⁴Axtra XAP ® providedby Danisco A/S.

Bird weights by pen were recorded at study initiation, 23 d, 35 d, andtermination (42 d). The pen was the unit of measure. Broiler diets werefed as crumbles (starter) or pellets (grower and finisher). Diets met orexceeded NRC standards (Table 2). The mixer was flushed to prevent crosscontamination of diets. All treatment feeds were mixed using a DavisS-20 mixer and pelleted using a California Pellet Mill (cold pellettemperature 65-70 C). Samples were collected from each treatment dietfrom the beginning, middle, and end of each batch and blended togetherto confirm enzyme activities and Enviva Pro presence in feed.

TABLE 2 Experimental diet composition of Example 1. Ingredient (%)Starter Grower Finisher Maize 53.62 57.87 59.82 Maize DDGS 10.00 10.0010.00 Soybean Meal 49% CP 26.93 23.97 21.36 Ampro 55 5.00 5.00 5.00 Soyoil 2.07 0.91 1.74 Lysine 0.24 0.24 0.24 DL-methionine 0.21 0.19 0.18L-threonine 0.01 0.01 0.01 Salt 0.30 0.34 0.35 Limestone 1.04 1.07 0.94Dicalcium phosphate 0.26 0.11 0.02 Vitamin and trace mineral premix 0.330.33 0.33 Calculated Nutrient Composition (%) CP 22.60 21.50 20.39Energy, kcal/kg 3060 3025 3100 Digestible lysine 1.36 1.26 1.21Digestible methionine 0.58 0.61 0.53 Digestible threonine 0.83 0.83 0.80

Birds received feed ad-libitum appropriate to the treatment from day 0to 42. Enzymes and Enviva Pro were provided by Danisco in theappropriate mixtures and levels for all experimental treatments. Alldiets contained 500 FTU of E. coli phytase in the background. The penswere arranged within the facility to prevent direct contact in order toavoid contamination. A change from starter to grower occurred on day 23.Grower diet was replaced with the finisher diet on day 35. At each feedchange, feeders were removed from pens by block, weighed back, emptied,and refilled with the appropriate treatment diet. On the final day ofthe study feed was weighed. Pens were checked daily for mortality. Whena bird was culled or found dead, the date and removal weight (kg) wererecorded. A gross necropsy was performed on all dead or culled birds todetermine the sex and probable cause of death. Signs of NecroticEnteritis were noted.

All pens had approximately 4 inches of built up litter with a coating offresh pine shavings. All birds were spray vaccinated prior to placementinto pens with a commercial coccidiosis vaccine (Coccivac-B). On days20, 21, and 22 all birds, except Treatment 1, were dosed with a brothculture of C. perfringens. A field isolate of C. perfringens known tocause NE and originating from a commercial broiler operation wasutilized as the challenge organism. Fresh inoculum was used each day.The titration levels were approximately 1.0×10⁸⁻⁹. Each pen received thesame amount of inoculum. The inoculum was administered by mixing intothe feed found in the base of the tube feeder. On day 23, five birdsfrom each pen were selected, euthanized, group weighed, and examined forthe degree of presence of Necrotic Enteritis lesions. The scoring wasbased on a 0 to 3 score, with 0 being normal and 3 being the most severe(0=none, 1=mild, 2=moderate, 3=marked/severe; Hofacre et al., 2003 J.Appl. Poult. Res. 12:60-64). No concomitant drug therapy was used duringthe study.

Means were separated using pair wise t-tests. Significant differenceswere considered at P<0.05. Pens were used as the experimental unit.

Results

FIG. 1 shows the necrotic enteritis lesion scores of broiler chickens ina necrotic enteritis challenge model, based on a 0 to 3 score system.Pooled SEM=0.15

The challenged control treatment increased lesion scores compared to theunchallenged control treatment. Addition of DFMs with a combination of axylanase, amylase, protease and phytase reduced lesion scores comparedto all other treatments. Addition of DFMs in combination with theenzymes reduced lesion scores compared DFMs alone or enzymes bythemselves.

FIG. 2 shows the body weight gain of broiler chickens in a necroticenteritis challenge model. Pooled SEM=28.6

FIG. 2 shows that a combination of the DFM (Enviva Pro®) with acombination of a xylanase, an amylase, a protease and a phytasesignificantly improved body weight gain (BW gain) in broiler chickenschallenged with Clostridium perfringens compared with the challengedcontrol—even resulting in BW gain which was improved over a negativecontrol (i.e. an unchallenged control). This was significantly betterthan any other treatments.

FIG. 3 shows the feed conversion ratio of broiler chickens in a necroticenteritis challenge model. Pooled SEM=0.016

The combination of Enviva Pro (DFM) with a xylanase, amylase, proteaseand phytase significantly improved (reduced) FCR (g BW gain/g feedintake) of broilers from hatch to 42 d compared to the challengedcontrol, and enzymes by themselves and the other treatments.

Example 2 Materials and Methods

Cobb 500 male broiler chicks were obtained from a commercial hatchery. Atotal of 26 chicks were randomly assigned to one of 8 replicate pens pertreatment. Floor pens (16 ft²/pen) were located in a curtain-sided housecontaining controlled heating, circulating fans, heat lamps and freshwood shavings. Birds were exposed to fluorescent lighting in a 24 hlight cycle for the first four days and then 16 light:8 hour dark cyclefor the remainder of the experiment. Feed was provided in bell feedersand water supplied via nipple drinkers ad libitum. A 5× dose ofCoccivac-B (Intervet) was administered manually with a syringe into theoral cavity of chicks at one day of age.

TABLE 3 Experimental design of Example 2. Coccidiosis AdditionalTreatment vaccine Phytase¹ enzyme² DFM³ 1 5X 500 FTU/kg None None 2 5X500 FTU/kg None Enviva Pro (7.5 × 10⁴ CFU/g) 3 5X 500 FTU/kg Xylanase⁴None (1000 u/kg) Amylase⁴ (1800 u/kg) Protease⁴ (5000 u/kg) 4 5X 500FTU/kg Xylanase⁴ Enviva Pro (1000 u/kg) (7.5 × 10⁴ CFU/g) Amylase⁴ (1800u/kg)  Protease⁴ (5000 u/kg) ¹Phytase from E. coli. ²Amylase fromBacillus amyloliquefaciens, xylanase from Trichoderma reesei, proteasefrom Bacillus subtilis. ³Enviva Pro ® is combination of Bacillussubtilis strains Bs2084, LSSAO1 and 15AP4, provided by Danisco A/S.⁴Avizyme 1505XAP ® provided by Danisco A/S.

Chicks were fed diets with or without either Enviva Pro or xylanase,amylase, and protease (Avizyme 1502; Table 3). Enzymes and Enviva Prowere provided by Danisco in the appropriate mixtures and levels for allexperimental treatments. All diets contained 500 FTU of E. coli phytase.The pens were arranged within the facility to prevent direct contact inorder to avoid contamination.

All diets were corn-soybean meal-DDGS based diets. Starter diets wereprovided during the study (d1-20). Diets were pelleted (65-70° C.) andcrumbled. Samples were collected from each treatment diet from thebeginning, middle, and end of each batch and blended together to confirmenzyme activities and Enviva Pro presence in feed.

TABLE 4 Experimental diet composition of Example 2. Starter GrowerFinisher Ingredient (%) (0-20 d) (20-38 d) (38-48 d) Maize 50.60 52.357.40 Wheat Middilings 1.33 1.03 1.32 Maize DDGS 7.00 7.00 7.00 SoybeanMeal 34.60 33.50 28.60 Vegetable fat 2.50 2.50 2.50 Limestone 1.41 1.381.09 MD-Phosphate 1.20 1.00 0.84 DL- methionine 0.31 0.27 0.27 Salt 0.460.46 0.46 L-Lysine 0.29 0.23 0.28 Vitamin and Trace Mineral Premix 1.501.50 1.50 Calculated Nutrient Composition (%) ME poultry, kcal/kg 29503000 3040 CP 23.0 22.5 20.4 Calcium 0.85 0.81 0.75 Av. Phosphorus 0.380.35 0.32 TSAA 0.98 0.94 0.89 Lysine 1.36 1.29 1.20 Methionine 0.62 0.590.56

Body weights and feeder weights were recorded on day 1, 11, 20, 38 and48 for calculation of feed intake, body weight gain and feed conversion.Mortality and culls were monitored on a daily basis and used to adjustfor feed consumption and gain. One bird from six replicate pens waseuthanized by cervical dislocation for collection of mucosal scrapingson days 11 and 20. Mucosal scrapings were collected from the ileum(Meckel's diverticulum to the ileo-cecal junction). The ileum wasexcised and cut along its length to expose the lumen and then flushedquickly and gently with PBS to remove digesta. The edge of a microscopeslide was used to remove the mucosal layer by scraping along the lengthof the excised tissue section. The mucosal layer was immediately freezeclamped between aluminium plates in liquid N to preserve RNA integrityand stored in individual whirl-pack bags. Frozen tissue samples werestored in liquid N during sampling and at −80 C prior to analysis. TotalRNA from mucosal scraping was isolated using the Trizol reagent(Invitrogen) using a mechanical homogenizer for tissue disruption. TotalRNA (0.5 μg) was reverse transcribed to complementary DNA using iScript(Bio-Rad) according to the manufacturer's recommendations. The mRNAabundance of secreted inflammatory cytokine genes (interleukin-10,interferon-γ and interleukin-17) was assessed using chicken-specificprimers. Additionally, TATA-BP, HPRT-1 and β-actin mRNA abundance wasmeasured for data normalization using geNorm software. The fold-changein mRNA abundance in gene expression was determined using the modifieddelta-delta Ct equation as described by Rudrappa and Humphrey (2007) J.Nutr. 137: 427-432 and log transformed for data analysis.

Means were separated using pair wise t-tests. Significant differenceswere considered at P<0.05. Birds were used as the experimental unit formRNA data.

Results

FIG. 4 shows mRNA abundance of interferon-gamma gene in ileal mucosalscrapings of broiler chickens.

Age 11 d: Pooled SEM=0.1 Age 20 d: Pooled SEM=0.6

The combination of Enviva Pro and xylanase, amylase, protease+phytaseupregulated IFR-g expression in the ileum of 11-d-old-broilers thatreceived 5 times a live coccidiosis vaccine at hatch compared to thenegative control, Enviva Pro+phytase, and xylanase, amylase,protease+phytase. At 21 d, Enviva Pro+phytase, and the combination ofEnviva Pro and xylanase, amylase, protease+phytase down regulated IFR-gexpression in the ileum compared to the negative control. These datasuggest that modulation of immune response may be one of the mechanismsof improved performance of DFMs in combination with the 4 enzymes inbroilers.

FIG. 15 shows feed conversion ratio of broiler chickens at 48 d of age.Age 48 d: Pooled SEM=0.041

Example 3 Materials and Methods

One digestibility trial with broiler chickens was conducted to determinethe effects of dietary enzymes and DFMs treatments on nutrientutilisation. The cages were housed in environmentally controlled rooms.The birds received 20-hour fluorescent illumination and, allowed freeaccess to the diets and water. On day 1, a broiler live coccidiosisvaccine was given to all chicks via drinking water. Paper was providedon cage wire-floor for the first three days to enable recycling ofEimeria Oocystes. The study consisted of the following treatments (Table5).

TABLE 5 Experimental design of Example 3. Treatment Phytase¹ Additionalenzyme² DFM³ 1 500 FTU/kg None None 2 500 FTU/kg Xylanase⁴ (1000 u/kg)None Amylase 1⁴ (1800 u/kg) Protease⁴ (5000 u/kg) 3 500 FTU/kg Xylanase(2000 u/kg) None Amylase 2 (200 u/kg) 4 500 FTU/kg Xylanase⁵ (2000 u/kg)None Amylase 2⁵ (200 u/kg) Protease⁵ (5000 u/kg) 5 500 FTU/kg NoneEnviva Pro (7.5 × 10⁴ CFU/g) 6 500 FTU/kg Xylanase⁴ (1000 u/kg) EnvivaPro Amylase 1⁴ (1800 u/kg) (7.5 × 10⁴ CFU/g) Protease⁴ (5000 u/kg) 7 500FTU/kg Xylanase (2000 u/kg) Enviva Pro Amylase 2 (200 u/kg) (7.5 × 10⁴CFU/g) 8 500 FTU/kg Xylanase⁵ (2000 u/kg) Enviva Pro Amylase 2⁵ (200u/kg) (7.5 × 10⁴ CFU/g) Protease⁵ (5000 u/kg) ¹Phytase from E. coli.²Amylase 1 from Bacillus amyloliquefaciens, amylase 2 from Bacilluslicheniformis, xylanase from Trichoderma reesei, protease from Bacillussubtilis. ³Enviva Pro ® is combination of Bacillus subtilis strainsBs2084, LSSAO1 and 15AP4, provided by Danisco A/S. ⁴Avizyme 1505 ®provided by Danisco A/S. ⁵Axtra XAP ® provided by Danisco A/S.

A total of 192 birds were individually weighed and assigned on the basisof body weight to 48 cages (4 birds/cage). The 8 dietary treatments werethen randomly assigned to six cages each. Birds received starter feedad-libitum appropriate to the treatment from 0 to 21 days. Enzymes andEnviva Pro were provided by Danisco in the appropriate mixtures andlevels for all experimental treatments. All diets contained 500 FTU ofE. coli phytase. The pens were arranged within the facility to preventdirect contact in order to avoid contamination. Birds were fed starterdiets (Table 6) in mash form throughout the experiment.

TABLE 6 Experimental diet composition of Example 3. Ingredient (%)Starter Maize 46.22 Wheat middlings 6.73 Maize DDGS 7.00 Soybean Meal48% CP 32.81 Maize starch/enzyme/DFM premix 0.30 Animal/vegetable fatblend (50:50) 3.00 L-Lysine•HCl 0.27 DL-methionine 0.30 L-threonine 0.11Titanium dioxide 0.30 Salt 0.34 Limestone 1.12 Dicalcium phosphate 1.20Vitamin and trace mineral premix 0.30 Calculated Nutrient Composition(%) CP 23.00 ME, kcal/kg 2950 Calcium 0.85 Available phosphorus 0.38Sodium 0.18 Digestible lysine 1.21 Digestible methionine 0.62 DigestibleTSAA 0.86 Digestible threonine 0.76

On day 21, four birds per cage were euthanized by intracardial injectionof sodium pentobarbitone and contents of the lower ileum were expressedby gentle flushing with distilled water. Digesta from birds within acage were pooled, resulting in six samples per dietary treatment. Thedigesta samples were frozen immediately after collection, lyophilisedand processed. Digesta samples and diets were analysed for Ti, DM, GE,starch, fat, N and amino acids, excluding tryptophan, as per standardprocedures. Calculation of ileal digestibility coefficients wasperformed as reported by Ravindran et al. (2005), based on theconcentration of indigestible Ti. The energy contribution of starch, fatand protein to ileal digestible energy was calculated based on meangross energy of starch (4.2 kcal/g), fat (9.4 kcal/g), or protein (5.5kcal/kg). The improvement of digestible amino acids in response toenzymes and DFMs was expressed in relation to the amount of non-digestedamino acids at the ileal level; the slope of that linear function wasused as an indicator of the effects of the additives on amino aciddigestibility.

Means were separated using pair wise t-tests. Significant differenceswere considered at P<0.05. Cages were used as the experimental unit.

Results

FIG. 5 shows apparent ileal digestible energy of broiler chickens at 21d of age. Pooled SEM=0.027

The addition of Enviva Pro (a DFM) in combination with an amylase,xylanase, protease and phytase exhibited commercially relevantincrements of ileal digestible energy compared with the enzymes bythemselves and the negative controls. These data indicates that DFMsimproved the effects of these exogenous enzymes on the energydigestibility of poultry diets. For the avoidance of doubt Amylase 2 isthrough use of the amylase in AxtraXAP and Amylase 1 is through use ofthe amylase in Avizyme 1502.

FIG. 6 show increments of ileal amino acid digestibility for threedietary treatments versus the control treatment as function of ilealundigested amino acids in the control treatment using 21-d-old broilerchickens.

The figure presents the improvement on ileal amino acid digestibility ofdietary treatments with respect to the undigested fraction of aminoacids in the ileum of broilers in the control treatment. Each pointwithin a treatment represents one of the measured amino acids. Theaddition of Enviva Pro on top of xylanase, amylase 2, protease+phytaseincreased the ileal digestibility of amino acids (+11.3%) compared toEnviva Pro+Phytase (+3.6%) and xylanase, amylase 2, protease+phytase bythemselves (i.e. without DFM) (+4.7%). These data indicates that DFMsimproved the efficacy of these exogenous enzymes to increase amino aciddigestibility of poultry diets.

FIG. 7 shows the improvement of ileal digestible energy with respect tothe control treatment using 21-d-old broiler chickens.

The figure presents the increment of ileal digestible energy of eachdietary treatment compared a negative control treatment with phytase.Additionally, the calculated contributions of energy from starch, fat orprotein are presented. Addition of Enviva Pro in combination withxylanase, amylase 2, protease+phytase increased the ileal digestibleenergy compared to the Enviva Pro+phytase treatment and the xylanase,amylase 2, protease+phytase by themselves treatment. Addition of EnvivaPro in combination with xylanase, amylase 1, protease+phytase producedcommercially important increments on ileal digestible energy versus theenzymes by themselves. These data indicate an improved ability of the 4enzymes to increase the ileal digestible energy of broiler diets in thepresence of DFMs.

Example 4 Materials and Methods

One digestibility trial with broiler chickens was conducted to determinethe effects of dietary enzymes and DFMs treatments on nutrientutilisation. The cages were housed in environmentally controlled rooms.The birds received 20-hour fluorescent illumination and, allowed freeaccess to the diets and water. On day 1, a broiler live coccidiosisvaccine was given to all chicks via drinking water. Paper was providedon cage wire-floor for the first three days to enable recycling ofEimeria Oocystes. The study consisted of the following treatments (Table7)

TABLE 7 Experimental design of Example 4. Treatment Phytase¹ Additionalenzyme² DFM³ 1 None None None 2 500 FTU/kg Xylanase⁴ (1000 u/kg) NoneAmylase 1⁴ (1800 u/kg) Protease⁴ (5000 u/kg) 3 500 FTU/kg Xylanase⁵(2000 u/kg) None Amylase 2⁵ (200 u/kg) Protease⁵ (5000 u/kg) 4 None NoneEnviva Pro (7.5 × 10⁴ CFU/g) 5 500 FTU/kg Xylanase⁴ (1000 u/kg) EnvivaPro Amylase 1⁴ (1800 u/kg) (7.5 × 10⁴ CFU/g) Protease⁴ (5000 u/kg) 6 500FTU/kg Xylanase⁵ (2000 u/kg) Enviva Pro Amylase 2⁵ (200 u/kg) (7.5 × 10⁴CFU/g) Protease⁵ (5000 u/kg) ¹Phytase from E. coli. ²Amylase 1 fromBacillus amyloliquefaciens, amylase 2 from Bacillus licheniformis,xylanase from Trichoderma reesei, protease from Bacillus subtilis.³Enviva Pro ® is combination of Bacillus subtilis strains Bs2084, LSSAO1and 15AP4, provided by Danisco A/S. ⁴Avizyme 1505 ® provided by DaniscoA/S. ⁵Axtra XAP ® provided by Danisco A/S.

A total of 144 birds were individually weighed and assigned on the basisof body weight to 36 cages (4 birds/cage). The 6 dietary treatments werethen randomly assigned to six cages each. Birds received starter feedad-libitum appropriate to the treatment from 0 to 21 days. Enzymes andEnviva Pro were provided by Danisco in the appropriate mixtures andlevels for all experimental treatments. The pens were arranged withinthe facility to prevent direct contact in order to avoid contamination.Birds were fed starter diets (Table 6) in mash form throughout theexperiment.

TABLE 8 Experimental diet composition of Example 4. Ingredient (%)Starter Maize 46.22 Wheat middlings 6.73 Maize DDGS 7.00 Soybean Meal48% CP 32.81 Maize starch/enzyme/DFM premix 0.30 Animal/vegetable fatblend (50:50) 3.00 L-Lysine•HCl 0.27 DL-methionine 0.30 L-threonine 0.11Titanium dioxide 0.30 Salt 0.34 Limestone 1.12 Dicalcium phosphate 1.20Vitamin and trace mineral premix 0.30 Calculated Nutrient Composition(%) CP 23.00 ME, kcal/kg 2950 Calcium 0.85 Available phosphorus 0.38Sodium 0.18 Digestible lysine 1.21 Digestible methionine 0.62 DigestibleTSAA 0.86 Digestible threonine 0.76

Feed intake and total excreta output were measured quantitatively percage over four consecutive days (from day 17 to 20) for thedetermination of nitrogen-corrected apparent metabolizable energy (AMEn)and Nitrogen retention. Daily excreta collections were pooled within acage, mixed in a blender and sub-sampled. Each sub sample waslyophilized, ground to pass through a 0.5 mm sieve and stored inairtight plastic containers at −4 C pending analysis. Processed sampleswere analysed for DM, GE and N, using standard procedures.

Means were separated using pair wise t-tests. Significant differenceswere considered at P<0.05. Cages were used as the experimental unit.

Results

FIG. 8 shows nitrogen-corrected apparent metabolizable energy AMEn ofdietary treatments fed to 17 to 21-d-old broiler chickens. PooledSEM=0.015

Addition of Enviva Pro in combinations with xylanase, amylase,protease+phytase increased the AMEn of diets in response to enzymescompared to the negative control diet. In particular, addition of EnvivaPro in combination with xylanase, amylase 2, protease+phytase increasedthe AMEn of diets in response to enzymes compared to diets with onlyEnviva Pro.

FIG. 9 shows nitrogen retention of 17 to 21-d-old broiler chickens.Pooled SEM=0.006

Addition of Enviva Pro in combination with xylanase, amylase,protease+phytase increased the nitrogen retention of broiler chickens inresponse to enzymes compared to the negative control diet. Inparticular, addition of Enviva Pro on top of xylanase, amylase 2,protease+phytase increased the nitrogen retention of broilers inresponse to enzymes compared to broilers fed diets with Enviva Pro only.

Example 5 Materials and Methods

Ross 308 male broiler chicks were obtained from a commercial hatchery. Atotal of 10 chicks were randomly assigned to one of 6 replicate cagesper treatment. Birds were exposed to fluorescent lighting in a 24 hlight cycle for the first four days and then 16 light:8 hour dark cyclefor the remainder of the experiment. Feed and water were supplied adlibitum. The experimental design consisted of the following treatments.

TABLE 9 Experimental design of Example 5. Coccidiosis CoccidioAdditional Treatment vaccine stat Phytase¹ enzyme² DFM³ 1 None None 500FTU/kg None None 2 5X None 500 FTU/kg None None 3 5X Salinomycin 500FTU/kg None None 4 5X None 500 FTU/kg None Enviva Pro (7.5 × 10⁴ CFU/g)5 5X None 500 FTU/kg Xylanase⁴ (1000 u/kg) None Amylase⁴ (1800 u/kg)Protease⁴ (5000 u/kg) 6 5X None 500 FTU/kg Xylanase⁴ (1000 u/kg) EnvivaPro Amylase⁴ (1800 u/kg) (7.5 × 10⁴ CFU/g) Protease⁴ (5000 u/kg)¹Phytase from E. coli. ²Amylase from Bacillus amyloliquefaciens,xylanase from Trichoderma reesei, protease from Bacillus subtilis.³Enviva Pro ® is combination of Bacillus subtilis strains Bs2084, LSSAO1and 15AP4, provided by Danisco A/S. ⁴Avizyme 1505 ® provided by DaniscoA/S.

In treatments 2 to 6, an overdosed (recommended dose ×5) coccidiosisvaccine (B, Intervet) was administered manually with a syringe into theoral cavity of chicks at one day of age. In treatment 2, Salinomycin(Bio-cox) was used at the approved level (60 g/MT) as a coccidiostat.The pens were arranged within the facility to prevent direct contact inorder to avoid cross contamination with Eimeria oocysts and DFMs.Enzymes and Enviva Pro were provided by Danisco A/S in the appropriatemixtures and levels for all experimental treatments. All diets contained500 FTU of E. coli phytase in the background.

TABLE 10 Experimental diet composition of Example 5. Ingredient (%)Starter Maize 53.18 Maize DDGS 10.00 Soyabean Meal 48% CP 32.05 SoyabeanOil 1.07 L-Lysine HCl 0.31 DL-methionine 0.31 L-threonine 0.12 Salt 0.33Limestone 1.14 Dicalcium Phosphate 1.19 Vitamin and Trace Mineral Premix0.30 Calculated Nutrient Composition (%) CP 23.00 ME, kcal/kg 2950Calcium 0.85 Available phosphorus 0.38 Sodium 0.18 Digestible lysine1.21 Digestible methionine 0.63 Digestible TSAA 0.86 Digestiblethreonine 0.76

A total of 2 birds per replicate cage were euthanized at 14 d of age forcollection of mucosal scrapings from mid-ileum. Ileums were flushed withdistilled water and cut open with a pair of scissors. Opened sectionswere laid flat on a clean glass plate. Mucosa was carefully scraped fromthe mid region of ileum with the long edge of a glass slide. Each samplewas stored in 2 ml of RNA later (Ambion) and frozen in a −80 C freezer.Samples were thawed on ice. Total RNA was isolated with Trizol reagentaccording to standard protocols. Integrity of RNA was determined on anagarose gel. RNA was reverse transcribed with the MMLV reversetranscriptase. Expression of mucin (MUC2) was determined by real timePCR on a Biorad real-time MyIQ machine.

Means were separated using pair wise t-tests. Significant differenceswere considered at P<0.05. Birds were used as the experimental unit formRNA data.

Results

FIG. 10 shows mRNA abundance of MUC2 gene in ileal mucosal scrapings ofbroiler chickens at 14 d of age. Pooled SEM=0.14

Addition of Enviva Pro in combination with xylanase, amylase,protease+phytase down regulated the expression of MUC 2 in the ileum ofbroilers challenged with a 5× dose of a live coccidiosis vaccinecompared to the challenged control. These data suggest that a reductionof endogenous amino acid losses due to reduced mucin secretion may beresponsible for improved performance of broilers receiving combinationsof DFMs and the 4 enzymes.

Example 6 Materials and Methods

Tissue samples were taken from broiler chicks from the trial presentedin Example 1 at 23 days of age. Treatment specifications are presentedin Table 1. The jejunum, pancreas and liver were removed from 2 birdsfrom every pen and the mucosa pooled resulting in eight samples pertreatment. The samples were rinsed in buffer solution (PBS) immersed ina tissue storage reagent (RNAlater) according to manufacturer's protocoland stored at −80° C. Total RNA was isolated from each tissue sampleusing a single step phenol-chloroform extraction method as described byChomczynski and Saachi (1987; Anal. Biochem. 162:156-9). Concentrationof the RNA was determined by measuring the absorbance at 260 nm(Nanodrop) and monitored for integrity by gel electrophoresis on 1.2%agarose gels. Only RNA of sufficient purity and having a ratio ofabsorption at 260 nm vs. 280 nm greater than 1.87 were considered foruse.

Microarrays were manufactured using 70 base pair oligo-nucleotides(Opereon Biotechnologies Inc) according to the protocol described byDruyan et al. (2008; Poult. Sci. 87:2418-29). The experimental design ofthe array was a complete interwoven loop design as described by Garosiet al. (2005; Br. J. Nutr. 93:425-32) which each sample is compareddirectly with the others in a multiple pair wise fashion allowing alltreatments to be compared. The samples were labelled according to themethod described by Druyan et al. (2008; Poult. Sci. 87:2418-29) in thatthat half the samples would be labelled with Cy3 and half with Cy5 whichare fluorescent dyes of cyanine. Hybridisation was carried out using thePronto Plus! Microarray Hybridisation Kit prior to the addition of Cy3and Cy5 labelled cDNA probes and covered with a clean glass coverslip(Lifterslip) and left to hybridise for 16 hours. The microarrays werethen scanned on a Scan Array Gx PLUS Microarray Scanner set to 65% laserpower to acquire images.

Total RNA from individual samples was reversed transcribed to producecDNA which was then used as a template for the qPCR amplifications asdescribed by Druyan et al. (2008; Poult. Sci. 87:2418-29). Thermocyclingparameters were optimised for each gene and each gene was amplifiedindependently in duplicate within a single instrument run.

Data files were generated from the scanned images of the microarrays butextracting the intensity raw data for each slide and dye combinationusing ScanAlyze Softare. Intensity data files were then analysed usingJMP Genomics including and initial log 2 transformation. Datanormalisation was performed using locally-weighted regression andsmoothing first within array and across all arrays. The resultingnormalised log 2 intensities were analysed using a mixed model ANOVA.

Mean intensities were compared using a threshold of significance basedon Bongerroni correction of P=0.05. For the complete array, includingall replicates, a mean by grid intensity was calculated for each geneusing the 3 side by side probes, resulting in a total of four replicatedmeans, one from each grid, per gene. Data for the Ct ratio from thesamples in duplicate (sample gene Ct:Sample GAPDH Ct) depending ontreatment were subjected to one way ANOVA.

Results

Expression data was collected using the microarray platform and a “heatmap” produced to visualise the data for the jejunum (FIG. 16) andpancreas (FIG. 17). Relative expression levels of six genes of interestwere converted to visual cues based on the scale seen in FIG. 16. Lowlyexpressed genes are marked with a minus sign (“−”), and highly expressedgenes are marked with a plus sign (“+”); whereas a greater grayintensity depicts a greater difference from the mean expression level ofthe treatments. The genes that were measured and their purportedfunctions are seen in Table 11. Real-time PCR was used to validate thegene expression shown in the heat map for sucrase-isomaltase (SI) andamylase 2A (AMY2a) and were highly correlated to the array data.

TABLE 11 Purported function of genes measured. Gene Identity FunctionPEPT1 Oligo-peptide transporter 1 Nutrient transport GCK GlucokinaseInitial step in glucose metabolism SI Sucrase isomaltase Glucosemetabolism ZO1 Tight Junction protein 1 Tight junction formation,intestinal integrity CD3d T- cell antigen CD3 T-cell marker AMY2AAmylase 2A Starch and sucrose metabolism

FIG. 16 shows a heat map of expression profiles of genes of interest forall treatments for jejunum at 23 days of age.

FIG. 17 shows a heat map of expression profile of chicken alpha amylasefor all treatments in pancreas at 23 days of age.

In FIGS. 16 and 17 the key is as follows:

Unchallenged control=Unchallenged Control+phytaseCC=Challenged Control+phytaseCC+Amylase=Challenged Control+phytase+amylaseCC+XAP=Challenged Control+phytase+xylanase+amylase+proteaseCC+EP=Challenged Control+phytase+Enviva ProCC+EP+Amylase=Challenged Control+phytase+amylase+Enviva ProCC+EP+XAP=Challenged Control+phytase+xylanase+amylase+protease+EnvivaPro

The expression of oligo-peptide transport 1 (PEPT1) was increased byxylanase+amylase+protease+phytase, and this was increased further whenin combination with Enviva Pro. PEPT1 is part of a peptide transportsystem and is responsible for the uptake of a wide range of di- andtri-peptides.

The expression of Glucokinase (GCK) was down-regulated by the challengedcontrol but the combination of amylase+phytase orxylanase+amylase+protease+phytase with Enviva Pro produced anup-regulation similar to the unchallenged control. The extent of theup-regulation was greater than when xylanase+amylase+protease+phytasewere used with Enviva Pro.

A similar pattern was also seen with sucrase iso-maltase (SI) where thecombination of Enviva Pro with amylase+phytase orxylanase+amylase+protease+phytase produced a greater up-regulation thanboth the challenged and unchallenged control. GCK is a key enzyme inglucose metabolism and SI is responsible for hydrolysis of sucrose andiso-maltose, and so has an important role in the digestion andabsorption of carbohydrates in animals.

Tight Junction protein 1 (ZO1) was most highly expressed in thechallenged control. A reduction was seen with the enzyme treatments buta greater down-regulation in expression was seen when Enviva Pro wasused and particularly so when in combination withxylanase+amylase+protease+phytase which produced a similar level ofdown-regulation as the non-challenged control. ZO1 is a protein that ison the cytoplasmic face of tight junctions, there are various roles forthis protein ranging from signal transduction for tight junctionassembly to stability of the tight junctions themselves.

The T-cell antigen CD3 (CD3D) was highly expressed in the challengedcontrol. The enzyme alone treatments did reduce expression somewhat butit was significantly down-regulated when in combination with Enviva Pro.The combination of xylanase+amylase+protease+phytase produced thelargest down-regulation of the enzyme treatments, and, when incombination with Enviva Pro, produced an even larger down-regulationclose to that seen for the unchallenged control. CD3D is a surfacemolecule found on T cells and plays an important role in signaltransduction during T-cell receptor engagement and is part of the T-cellreceptor/CD3 complex.

The alpha amylase (AMY2A) was highly expressed in the unchallenged andchallenged controls but the addition of amylase+phytase orxylanase+amylase+protease+phytase resulted in reduced expression, whichwas further reduced when Enviva Pro was used in combination,particularly for xylanase+amylase+protease+phytase. Chicken alphaamylase is mainly produced in the pancreas and has a major role instarch digestion.

Discussion

The increase in expression of the peptide transporter oligopeptidetransporter 1 (PEPT1) when xylanase+amylase+protease+phytase were given,particularly in combination with Enviva Pro, suggests increasedavailability of peptides and thus an increased requirement of peptidetransporters, which indicates a synergistic effect of enzymes and DFMsto increase the adsorption of peptides for the animal which allows forgreater growth. Animal performance results of Example 1 support thisconclusion. The increase in expression of glucokinase and sucraseisomerase with the combination of amylase+phytase, orxylanase+amylase+protease+phytase, and Enviva Pro suggests that therewas increased absorption of glucose, and increased availability ofsucrose and isomaltose in the brush border, which indicates a positiveinteraction between the enzyme and DFMs to increase carbohydrateabsorption in the small intestine and thus increase energy availabilityfrom the diet. The decrease of glucokinase expression for the challengedcontrol suggests that the Clostridium perfringens challenge causeddamage to the mucosa and that addition of Enviva Pro andxylanase+amylase+protease+phytase alleviated this.

The effect of Enviva Pro on reducing the expression of Tight junctionprotein 1 indicates lower requirement for protein turn over in theintestine, which may be related to a high intestinal integrity. Theincreased expression in the challenged control, however, suggests thatturnover/requirement of the protein was high due to failing intestinalintegrity possibly due to the coccidia and Clostridium perfringensinfections. The enzymes alone did have some effect on ameliorating thisbut the additive effect seen with Enviva Pro suggests a greater benefitfrom the combination. This indicates that Enviva Pro acts to increaseintestinal integrity and thus benefit the health of the animal.Increased intestinal integrity, and thus absorptive capacity, may be oneof the mechanisms by which the effectiveness of exogenous enzymes isincreased when a DFM is present.

The increased expression of T cell antigen CD3 d in the challengedcontrol indicates increased cell-mediated immune response due to thechallenge. In these conditions, birds will be undergoing sub-optimalperformance because the immune response will demand energy that could beused for growth, and because some birds will experience a systemicdisease response. The increased expression of this immunological markerwas markedly reversed when Enviva Pro was used alone or in combinationwith enzymes. Down regulation of immune response in the intestine may beone of the mechanisms by which the effectiveness of exogenous enzymes innutrient absorption and performance is increased when a DFM is present.

The down-regulation of alpha amylase (AMY2A) production that was seenwith the combination of amylase+phytase, orxylanase+amylase+protease+phytase suggests that the chicken is reducingits production of endogenous amylase as a response to the exogenousenzymes supplied. The additive effect seen with Enviva Pro andxylanase+amylase+protease+phytase suggest that the DFM is workingsynergistically with the exogenous enzymes to allow the bird to utilisethe energy that it would have spent producing enzymes for digestion ofstarch in the diet.

The net effect of a down-regulated immune response and higher intestinalintegrity, and a better nutrient digestion and absorption with thecombination of enzymes and DFMs, clearly determines enhanced productionperformance of broiler chickens.

Example 7 Materials and Methods

A digestibility trial with broiler chickens was conducted to determinethe effects of dietary enzymes and DFM treatments on energy utilisation.A total of 288 day-old, male Ross 308 chicks were obtained from acommercial hatchery and brooded in raised wire battery pens until day14. Birds were vaccinated with a live coccidia vaccine at hatch(Coccivac-B). Chicks were fed a corn-SBM-DDGS based starter diet. Chickswere provided experimental diets from day 14 until day 21. The feed andwater were provided ad-libitum throughout the 21 day period. Six chickswere housed per pen in battery pens located within an environmentallycontrolled room, where they received supplemental heat starting at 35°C. on day-of-age and decreasing 2° C. weekly. Light was provided at23L:1D. On day 15, chicks were individually weighed, sorted, wing bandedand randomly allocated to the experimental units using a completelyrandomized design. Each treatment consisted of 8 pens per treatment. Thestudy consisted of the following treatments (Table 12).

TABLE 12 Experimental design of Example 7. Treatment Phytase¹ Additionalenzyme² DFM^(3, 4) 1 None None None 2 500 Xylanase (2000 u/kg) NoneFTU/kg Amylase (200 u/kg) Protease (5000 u/kg) 3 None None Enviva Pro(1.5 × 10⁵ FTU/g) 4 500 Xylanase (2000 u/kg) Enviva Pro FTU/kg Amylase(200 u/kg) (1.5 × 10⁵ FTU/g) Protease (5000 u/kg) 5 None None GalliProTect (8 × 10⁵ FTU/g) 6 500 Xylanase (2000 u/kg) GalliPro Tect FTU/kgAmylase (200 u/kg) (8 × 10⁵ FTU/g) Protease (5000 u/kg) ¹Phytase fromButtiauxella. ²Amylase from Bacillus licheniformis, xylanase fromTrichoderma reesei, protease from Bacillus subtilis. ³Enviva Pro ® is acombination of Bacillus subtilis strains Bs2084, LSSAO1 and 15AP4,provided by Danisco A/S. ⁴GalliPro Tect is a DFM comprised by one strainof Bacillus licheniformis (DSM17236).

Enzymes and DFMs were sourced and provided by Danisco in the appropriatemixtures and levels for all experimental treatments. The pens werearranged within the facility to prevent direct contact in order to avoidcross contamination. Birds were fed starter diets (Table 13) in mashform throughout the experimental period.

TABLE 13 Experimental diet composition of Example 7. Ingredient (%)Starter Corn 52.94 Corn-DDGS 12.00 Soybean meal 48% 29.38Animal/Vegetable Fat Blend 1.08 Salt 0.40 DL Methionine 0.22 Bio-Lys0.44 Limestone 1.30 Dicalcium Phosphate 1.27 Choline chloride 60 0.10Vit/Min Premix 0.63 TiO₂ 0.25 Calculated Nutrient Composition (%) CP22.25 ME, kcal/kg 2925 Calcium 0.90 Available phosphorus 0.38 Sodium0.18 Digestible lysine 1.20 Digestible methionine 0.52 Digestible TSAA0.85 Digestible threonine 0.75

Clean excreta trays were put in place for the last 2 days and excretasamples were collected by pen on day 21. The collected excreta sampleswere frozen at −20° C. before they were oven dried at 65° C. for 3 daysto determine the dry matter (AOAC International, 2005; method 934.01).The feed samples were also corrected to the dry matter basis bemeasuring 5.0 g of each sample and drying them in an oven at 100° C. for24 hrs. The excreta samples were then ground through a 1-mm screen whilethe feed samples were ground 0.5-mm screen. Excreta samples and dietswere analysed for Ti, DM, GE, and N, as per standard procedures.Apparent metabolizable energy (AME) calculation was based on theconcentration of the indigestible marker (Ti) and the gross energy ofdiets and excreta. Appropriate corrections were made for differences inmoisture content. N-corrected AME (AMEn) was determined for zeronitrogen retention by multiplication with 8.22 kcal per gram of nitrogenretained in the body (Hill and Anderson, 1958; J. Nutr. 64:587-603).

Means were separated using pair wise t-tests. Significant differenceswere considered at P<0.05. Cages were used as the experimental unit.

Results

FIG. 18 shows apparent metabolizable energy corrected by nitrogenretention (AME_(n)) of 21 d old broiler chickens. Effect of DFM;P<0.001; Effect of Enzyme; P<0.001; Effect of DFM×Enzyme; P=0.27; PooledSEM=32 kcal.

Addition of Xylanase, amylase, protease, and phytase, in combinationwith Enviva Pro or GalliPro Tect resulted in improvements of AME_(n)versus the control treatment, that were significantly greater comparedto the enzymes or the DFMs by themselves. The AME_(n) increments due tothe combination of xylanase, amylase, protease, phytase, and Enviva Pro(235 kcal/kg) or GalliPro Tect (215 kcal/kg) were greater than theaddition of the enzymes and the DFM effects when applied separately (152kcal/kg for Enviva Pro, or 120 kcal/kg for GalliPro Tect), compared tothe negative control treatment.

Example 8 Materials and Methods

One thousand and four hundred one-day-old Cobb male chicks werepurchased from a commercial hatchery. At study initiation, fifty maleswere allocated to one of seven pens per treatment by blocks. The studyconsisted of the following treatments (Table 1):

TABLE 1 Experimental design of Example 8. Clostridium perfringensAdditional Treatment Challenge Phytase¹ enzyme² DFM³ 1 No 500 None NoneFTU/kg 2 Yes 500 None None FTU/kg 3 Yes 500 None Enviva Pro FTU/kg (7.5× 10⁴ FTU/g) 4 Yes 500 Xylanase⁴ Enviva Pro FTU/kg (2000 u/kg) (7.5 ×10⁴ FTU/g) Amylase⁴ (200 u/kg)  Protease⁴ (5000 u/kg) ¹Phytase from E.coli. ²Amylase from Bacillus licheniformis, xylanase from Trichodermareesei, protease from Bacillus subtilis. ³Enviva Pro ® is combination ofBacillus subtilis strains Bs2084, LSSAO1 and 15AP4, provided by DaniscoA/S. ⁴Axtra XAP ® provided by Danisco A/S.

Bird weights by pen were recorded at study initiation, 21 d andtermination (42 d). The pen was the unit of measure. Broiler diets werefed as crumbles (starter) or pellets (grower and finisher). Diets met orexceeded NRC standards (Table 2). The mixer was flushed to prevent crosscontamination of diets. All treatment feeds were mixed using a DavisS-20 mixer and pelleted using a California Pellet Mill (cold pellettemperature 65-70 C). Samples were collected from each treatment dietfrom the beginning, middle, and end of each batch and blended togetherto confirm enzyme activities and Enviva Pro presence in feed.

TABLE 2 Experimental diet composition of Example 8. Ingredient (%)Starter Grower Finisher Maize 50.959 59.6156 62.7488 Maize DDGS 12 12 12Soybean Meal 49% CP 30.7176 22.5873 19.4 Choline Chloride 0.06 0.06 0.06Soy oil 3.0693 2.7035 2.84841 Lysine 0.21 0.2426 0.244 DL-methionine0.1723 0.1566 0.1341 L-threonine 0.0387 0.0551 0.0564 Salt 0.4668 0.46920.47 Limestone 1.4467 1.4501 1.33389 Dicalcium phosphate 0.7346 0.53490.571 Vitamin and trace 0.125 0.125 0.125 mineral premix CalculatedNutrient Composition (%) CP 22.642 19.45 19.45 Energy, mcal/kg 12.76112.012 12.012 Digestible lysine 1.327 1.124778 1.124778 Digestiblemethionine 0.53142 0.475425 0.475425 Digestible threonine 0.894010.78494 0.78494

Birds received feed ad-libitum appropriate to the treatment from day 0to 42. Enzymes and Enviva Pro were provided by Danisco in theappropriate mixtures and levels for all experimental treatments. Alldiets contained 500 FTU of E. coli phytase in the background. The penswere arranged within the facility to prevent direct contact in order toavoid contamination.

A change from starter to grower occurred on day 21. Grower diet wasreplaced with the finisher diet on day 35. At each feed change, feederswere removed from pens by block, weighed back, emptied, and refilledwith the appropriate treatment diet. On the final day of the study, feedwas weighed. Pens were checked daily for mortality. When a bird wasculled or found dead, the date and removal weight (kg) were recorded. Agross necropsy was performed on all dead or culled birds to determinethe sex and probable cause of death. Signs of Necrotic Enteritis werenoted.

All pens had approximately 4 inches of built up litter with a coating offresh pine shavings. All birds were spray vaccinated prior to placementinto pens with a commercial coccidiosis vaccine (Coccivac-B). On days18, 19, and 20 all birds, except Treatment 1, were dosed with a brothculture of C. perfringens. A field isolate of C. perfringens known tocause Necrotic Enteritis and originating from a commercial broileroperation was utilized as the challenge organism. Fresh inoculum wasused each day. The titration levels were approximately 1.0×10⁸⁻⁹. Eachpen received the same amount of inoculum. The inoculum was administeredby mixing into the feed found in the base of the tube feeder.

Sample Collection

On day 21, a total of 8 birds per treatment (1-2 birds per pen) wereeuthanised and the total gastrointestinal tract from below the gizzardto the ileal-cecal junction was collected from each bird and sentovernight on ice to the laboratory. The samples were further dissectedin the laboratory to obtain a 20 cm portion of the jejunum surroundingthe Meckle's diverticulum; the remainder of the intestinal tract wasdiscarded. The sections were rinsed with 0.1% peptone to remove theintestinal contents and opened longitudinally to expose the epitheliallining. The sections were masticated in 99 ml of 0.1% peptone at 7.0strokes/s for 60 s to release mucosa-associated bacterial cells.Bacteria were harvested from the masticated solution by centrifugationat 12,000×g for 10 minutes. The resultant bacterial pellet wasresuspended in 10 ml of MRS broth+10% glycerol, flash-frozen in liquidnitrogen, and stored at −20° C. until further analysis.

DNA Isolation

Genomic DNA was isolated from all samples by phenol chloroformextraction and purified using Roche Applied Science High Pure PCRTemplate Purification Kit (Roche Diagnostics Corp., Indianapolis, Ind.).Samples were randomly pooled in pairs at the DNA level after extraction,for a total of four samples per treatment.

Pyrosequencing

Bacterial tag-encoded FLX amplicon pyrosequencing was performed asdescribed by Dowd (Dowd et al. 2008; BMC Microbiol. 8, 125). Anequivalent amount of DNA isolated from the intestinal mucosa from eachbird was analyzed in pooled samples containing DNA from two birds. TheV1-V3 region of the 16S rRNA gene was amplified in each sample using theprimers 28 F (5′-GAGTTTGATCNTGGCTCAG) and 519R (5′-GTNTTACNGCGGCKGCTG).Following sequencing, raw data was screened and trimmed based onquality. Sequences were sorted by individual samples based on barcodesequences. Barcode tags were removed and non-bacteria ribosomalsequences were removed. The bacterial community composition wasdetermined using BlastN comparison to a quality controlled and manuallycurated database derived from NCBI. The relative abundance of eachbacterial ID was determined for each sample. Data was compiled at eachtaxonomic level using NCBI nomenclature.

Statistical Analysis

For performance data means were separated using pair wise t-tests.Significant differences were considered at P<0.05. Pens were used as theexperimental unit.

Genus level identifications were used for the analysis of thepyrosequencing data. The relative abundance of each genus was calculatedand used for the analysis. The results were analysed using a categoricalmodel analysis and then a Chi-square probability calculated using JMP8.0.2 (SAS institute, Cary, N.C.), where each sample representing twobirds was considered an experimental unit.

Results:

FIG. 19 shows feed conversion ratio (FCR) of broiler chickens in anecrotic enteritis challenge model (Pooled SEM: 0.015).

The combination of Enviva Pro with xylanase, amylase, protease+phytasereduced FCR (g BW gain/g feed intake) compared to the challenged controltreatment and the use of Enviva Pro and phytase alone. Feed conversionratio was reduced by the combination to the level of the unchallengedcontrol+phytase.

FIG. 20 shows relative abundance of Lactobacillus spp. at 21 d in thejejunal mucosa of broiler chickens, ChSq<0.0001.

FIG. 20 shows the relative abundance of Lactobacillus spp. in comparisonto other species in the jejunal mucosa of broilers at 21 days in anecrotic enteritis challenge model. The proportion of Lactobacilli wasreduced in the challenged control in comparison to the unchallengedcontrol. The combination of Enviva Pro, xylanase, amylase,protease+phytase increases the proportion of Lactobacilli more so thanEnviva Pro and phytase alone and the challenged control.

Lactobacilli are widely used as probiotics for both human and animal use(Patterson and Burkeholder 2003; Poult Sci 82 (4) 627-31) and have beendocumented to improve gut health to a level that could be comparable toantibiotic growth promoters (Awad et al. 2009 Poult Sci 88 (1) 49-56).Thus by increasing the proportion Lactobacilli in the gut microbiota,the combination of Enviva Pro, xylanase, amylase, protease+phytase canimprove gut health and positively impact feed efficiency.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

1. A feed additive composition comprising a direct fed microbial incombination with a protease, a xylanase, an amylase and a phytase.
 2. Afeed additive composition according to claim 1 wherein the direct fedmicrobial is an antipathogen direct fed microbial.
 3. A feed additivecomposition according to claim 1 or claim 2 wherein the direct fedmicrobial is a viable bacterium.
 4. A feed additive compositionaccording to any one of claims 1 to 3 wherein the direct fed microbialcomprises a bacterium from one or more of the following genera:Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus,Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium,Bifidobacterium, Clostridium and Megasphaera and combinations thereof.5. A feed additive composition according to any one of the precedingclaims wherein the direct fed microbial comprises a bacterium from oneor more of the following species: Bacillus subtilis, Bacilluslicheniformis, Bacillus amyloliquefaciens, Enterococcus, Enterococcusspp, and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp,Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis,Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillusfarciminus, lactobacillus rhamnosus, Clostridium butyricum,Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Bacilluscereus, Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii,Propionibacteria sp and combinations thereof.
 6. A feed additivecomposition according to any one of the preceding claims wherein thedirect fed microbial is one or more of the following strains: Bacillussubtilis strains 3A-P4 (PTA-6506); 15A-P4 (PTA-6507); 22C-P1 (PTA-6508);2084 (NRRL B-500130); LSSA01 (NRRL-B-50104); BS27 (NRRL B-50105); BS 18(NRRL B-50633); and BS 278 (NRRL B-50634).
 7. A feed additivecomposition according to any one of the preceding claims wherein thedirect fed microbial is in the form of an endospore.
 8. A feed additivecomposition according any one of the preceding claims wherein thexylanase is an endo-1,4-β-d-xylanase or a 1,4 β-xylosidase preferably anendo-1,4-β-d-xylanase.
 9. A feed additive composition according to anyone of the preceding claims wherein the xylanase is from Bacillus,Trichoderma, Thermomyces, Aspergillus, Penicillium and Humicola.
 10. Afeed additive composition according to any one of the preceding claimswherein the protease is a subtilisin, a bacillolysin, an alkaline serineprotease, a keratinase or a Nocardiopsis protease.
 11. A feed additivecomposition according to any one of the preceding claims wherein thephytase is a 6-phytase or a 3-phytase.
 12. A feed additive compositionaccording to claim 11 wherein the phytase is a 6-phytase.
 13. A feedadditive composition according to any one of the preceding claimswherein the phytase is an E. coli phytase or a Buttiauxella phytase orHafnia phytase or a Citrobacter phytase or a Aspergillus phytase or aPenicillium phytase or a Trichoderma phytase or a Hansenula phytase. 14.A feed additive composition according to any one of the preceding claimswherein the amylase is selected from one or more of the group consistingof: an α-amylase, a G4-forming amylase, a β-amylase and a γ-amylases.15. A feed additive composition according to claim 14 wherein theamylase is an α-amylase.
 16. A feed additive composition according toany one of the preceding claims wherein the amylase is from Bacilluslicheniformis, B. amyloliquefaciens, Trichoderma spp. or Aspergillusspp.
 17. A feed additive composition according to any one of thepreceding claims wherein the phytase is present at a dosage of between200 FTU/g feed additive composition and 10000 FTU/g feed additivecomposition.
 18. A feed additive composition according to any one of thepreceding claims wherein the amylase is present at a dosage of between50 AU/g feed additive composition and 20000 AU/g feed additivecomposition.
 19. A feed additive composition according to any one of thepreceding claims wherein the xylanase is present at a dosage of between500 XU/g feed additive composition and 40000 XU/g feed additivecomposition.
 20. A feed additive composition according to any one of thepreceding claims wherein the protease is present at a dosage of 1000PU/g feed additive composition and 60000 PU/g feed additive composition.21. A feed additive composition according to any one of the precedingclaims wherein the DFM is present at a dosage of 3.75×10⁷ CFU/g feedadditive composition and 1×10¹¹ CFU/g feed additive composition.
 22. Amethod for improving the performance of a subject or for improvingdigestibility of a raw material in a feed (e.g. nutrient digestibility,such as amino acid digestibility), or for improving nitrogen retention,or for improving the subjects resistance to necrotic enteritis or forimproving feed conversion ratio (FCR) or for improving body weight gainin a subject or for improving feed efficiency in a subject or formodulating (e.g. improving) the immune response of the subject, or forpromoting the growth of beneficial bacteria in the gastrointestinaltract of a subject or for reducing populations of pathogenic bacteria inthe gastrointestinal tract of a subject, or for reducing nutrientexcretion in manure, which method comprising administering to a subjecta direct fed microbial in combination with a protease, a xylanase, anamylase and a phytase.
 23. A method according to claim 22 comprisingadministering the feed additive composition according to any one ofclaims 1-21.
 24. A method according to claim 22 or 23 wherein the directfed microbial is an antipathogen direct fed microbial.
 25. A methodaccording to any one of claims 22-24 wherein the direct fed microbial isa viable bacteria.
 26. A method according to any one of claims 22 to 25wherein the direct fed microbial comprises a bacterium from one or moreof the following genera: Lactobacillus, Lactococcus, Streptococcus,Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,Propionibacterium, Bifidobacterium, Clostridium and Megasphaera andcombinations thereof.
 27. A method according to any one of claims 22-26wherein the direct fed microbial comprises a bacterium from one or moreof the following species: Bacillus subtilis, Bacillus licheniformis,Bacillus amyloliquefaciens, Enterococcus, Enterococcus spp, andPediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillusacidophilus, Pediococsus acidilactici, Lactococcus lactis,Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillusfarciminus, lactobacillus rhamnosus, Clostridium butyricum,Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Bacilluscereus, Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii,Propionibacteria sp and combinations thereof.
 28. A method according toany one of claims 22-27 wherein the direct fed microbial is one or moreof the following strains: Bacillus subtilis strains 3A-P4 (PTA-6506);15A-P4 (PTA-6507); 22C-P1 (PTA-6508); 2084 (NRRL B-500130); LSSA01(NRRL-B-50104); BS27 (NRRL B-50105); BS 18 (NRRL B-50633); and BS 278(NRRL B-50634).
 29. A method according to any one of claims 22-28wherein the direct fed microbial is in the form of an endospore.
 30. Amethod according to any one of claims 22-29 wherein the xylanase is anendo-1,4-β-d-xylanase or a 1,4 β-xylosidase preferably anendo-1,4-β-d-xylanase.
 31. A method according to any one of claims 22-30wherein the xylanase is from Bacillus, Trichoderma, Thermomyces,Aspergillus, Penicillium and Humicola.
 32. A method according to any oneof claims 22-31 wherein the protease is a subtilisin, a bacillolysin, analkaline serine protease or a keratinase or a Nocardiopsis protease. 33.A method according to any one of claims 22-32 wherein the phytase is a6-phytase or a 3-phytase.
 34. A method according to claim 33 wherein thephytase is a 6-phytase.
 35. A method according to any one of claims22-34 wherein the phytase is an E. coli phytase or a Buttiauxellaphytase or Hafnia phytase or Citrobacter phytase or a Aspergillusphytase or a Penicillium phytase or a Trichoderma phytase or a Hansenulaphytase.
 36. A method according to any one of claims 22-35 wherein theamylase is selected from one or more of the group consisting of: anα-amylase, a G4-forming amylase, a β-amylase and a 7-amylases.
 37. Amethod according to any one of claims 22-36 wherein the amylase is anα-amylase.
 38. A method according to any one of claims 22-37 wherein theamylase is from Bacillus licheniformis, B. amyloliquefaciens,Trichoderma spp. or Aspergillus spp.
 39. Use of a direct fed microbialin combination with a protease, a xylanase, an amylase and a phytase forimproving the performance of a subject or for improving digestibility ofa raw material in a feed (e.g. nutrient digestibility, such as aminoacid digestibility) or for improving nitrogen retention) or forimproving the subject's resistance to of necrotic enteritis or forimproving feed conversion ratio (FCR) or for improving weight gain in asubject or for improving feed efficiency in a subject or for modulating(e.g. improving) the immune response of the subject or for promoting thegrowth of beneficial bacteria in the gastrointestinal tract of a subjector for reducing populations of pathogenic bacteria in thegastrointestinal tract of a subject, or for reducing nutrient excretionin manure.
 40. Use according to claim 39 wherein the feed additivecomposition according to any one of claims 1-21 is used.
 41. Useaccording to any one of claims 39-40 wherein the direct fed microbial isan antipathogen direct fed microbial.
 42. Use according to any one ofclaims 39-41 wherein the direct fed microbial is a viable bacterium. 43.Use according to any one of claims 39-42 wherein the direct fedmicrobial comprises a bacterium from one or more of the followinggenera: Lactobacillus, Lactococcus, Streptococcus, Bacillus,Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,Propionibacterium, Bifidobacterium, Clostridium and Megasphaera andcombinations thereof.
 44. Use according to any one of claims 39-43wherein the direct fed microbial comprises a bacterium from one or moreof the following species: Bacillus subtilis, Bacillus licheniformis,Bacillus amyloliquefaciens, Enterococcus, Enterococcus spp, andPediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillusacidophilus, Pediococsus acidilactici, Lactococcus lactis,Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillusfarciminus, lactobacillus rhamnosus, Clostridium butyricum,Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Bacilluscereus, Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii,Propionibacteria sp and combinations thereof.
 45. Use according to anyone of claims 39-44 wherein the direct fed microbial is one or more ofthe following strains: Bacillus subtilis strains 3A-P4 (PTA-6506);15A-P4 (PTA-6507); 22C-P1 (PTA-6508); 2084 (NRRL B-500130); LSSA01(NRRL-B-50104); BS27 (NRRL B-50105); BS 18 (NRRL B-50633); and BS 278(NRRL B-50634).
 46. Use according to any one of claims 39-45 wherein thedirect fed microbial is in the form of an endospore.
 47. Use accordingto any one of claims 39-46 wherein the xylanase is anendo-1,4-β-d-xylanase or a 1,4 β-xylosidase preferably anendo-1,4-β-d-xylanase.
 48. Use according to any one of claims 39-47wherein the xylanase is from Bacillus, Trichoderma, Thermomyces,Aspergillus, Penicillium and Humicola.
 49. Use according to any one ofclaims 39-48 wherein the protease is a subtilisin, a bacillolysin, analkaline serine protease or a keratinase or a Nocardiopsis protease. 50.Use according to any one of claims 39-49 wherein the phytase is a6-phytase or a 3-phytase.
 51. Use according to claim 50 wherein thephytase is a 6-phytase.
 52. Use according to any one of claims 39-51wherein the phytase is an E. coli phytase or a Buttiauxella phytase orHafnia phytase or Citrobacter phytase or a Aspergillus phytase or aPenicillium phytase or a Trichoderma phytase or a Hansenula phytase. 53.Use according to any one of claims 39-52 wherein the amylase is selectedfrom one or more of the group consisting of: an α-amylase, a G4-formingamylase, a β-amylase and a γ-amylases.
 54. Use according to any one ofclaims 39-53 wherein the amylase is an α-amylase.
 55. Use according toany one of claims 39-54 wherein the amylase is from Bacilluslicheniformis, B. amyloliquefaciens, Trichoderma spp. or Aspergillusspp.
 56. A kit comprising a direct fed microbial, a protease, axylanase, an amylase, a phytase and instructions for administration. 57.A kit according to claim 56 wherein said kit comprises the feed additivecomposition according to any one of claims 1-21.
 58. A kit according toany one of claims 56-57 wherein the direct fed microbial is anantipathogen direct fed microbial.
 59. A kit according to any one ofclaims 56-58 wherein the direct fed microbial is a viable bacterium. 60.A kit according to any one of claims 56-59 wherein the direct fedmicrobial comprises a bacterium from one or more of the followinggenera: Lactobacillus, Lactococcus, Streptococcus, Bacillus,Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,Propionibacterium, Bifidobacterium, Clostridium and Megasphaera andcombinations thereof.
 61. A kit according to any one of claims 56-60wherein the direct fed microbial comprises a bacterium from one or moreof the following species: Bacillus subtilis, Bacillus licheniformis,Bacillus amyloliquefaciens, Enterococcus, Enterococcus spp, andPediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillusacidophilus, Pediococsus acidilactici, Lactococcus lactis,Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillusfarciminus, lactobacillus rhamnosus, Clostridium butyricum,Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Bacilluscereus, Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii,Propionibacteria sp and combinations thereof.
 62. A kit according to anyone of claims 56-61 wherein the direct fed microbial is one or more ofthe following strains: Bacillus subtilis strains 3A-P4 (PTA-6506);15A-P4 (PTA-6507); 22C-P1 (PTA-6508); 2084 (NRRL B-500130); LSSA01(NRRL-B-50104); BS27 (NRRL B-50105); BS 18 (NRRL B-50633); and BS 278(NRRL B-50634).
 63. A kit according to any one of claims 56-62 whereinthe direct fed microbial is in the form of an endospore.
 64. A kitaccording to any one of claims 56-63 wherein the xylanase is anendo-1,4-β-d-xylanase or a 1,4 β-xylosidase preferably anendo-1,4-β-d-xylanase.
 65. A kit according to any one of claims 56-64wherein the xylanase is from Bacillus, Trichoderma, Thermomyces,Aspergillus, Penicillium and Humicola.
 66. A kit according to any one ofclaims 56-65 wherein the protease is a subtilisin, a bacillolysin, analkaline serine protease or a keratinase or a Nocardiopsis protease. 67.A kit according to any one of claims 56-66 wherein the phytase is a6-phytase or a 3-phytase.
 68. A kit according to claim 67 wherein thephytase is a 6-phytase.
 69. A kit according to any one of claims 56-68wherein the phytase is an E. coli phytase or a Buttiauxella phytase orHafnia phytase or Citrobacter phytase or an Aspergillus phytase or aPenicillium phytase or a Trichoderma phytase or a Hansenula phytase. 70.A kit according to any one of claims 56-69 wherein the amylase isselected from one or more of the group consisting of: an α-amylase, aG4-forming amylase, a β-amylase and a γ-amylases.
 71. A kit according toany one of claims 56-70 wherein the amylase is an α-amylase.
 72. A kitaccording to any one of claims 56-71 wherein the amylase is fromBacillus licheniformis or, B. amyloliquefaciens, Trichoderma spp. orAspergillus spp.
 73. A method of preparing a feed additive composition,comprising admixing a direct fed microbial with a protease, a xylanase,an amylase and a phytase and (optionally) packaging.
 74. A feedcomprising a feed additive composition according to any one of claims1-21.
 75. A feed according to claim 74 wherein the phytase is present ata dosage of between 400 FTU/kg feed and 1000 FTU/kg feed.
 76. A feedaccording to claim 74 or claim 75, wherein the amylase is present at adosage of between 100 AU/kg feed and 2000 AU/kg feed.
 77. A feedaccording to any one of claims 74-76 wherein the xylanase is present ata dosage of between 1000 XU/kg feed and 4000 XU/kg feed.
 78. A feedaccording to any one of claims 74-77 wherein the protease is present ata dosage of between 2000 PU/kg feed to 6000 PU/kg feed.
 79. A feedaccording to any one of claims 74-77 wherein the DFM is present at adosage of 7.5×10⁴ CFU/kg feed and 1×10⁷ CFU/kg feed.
 80. A method ofpreparing a feedstuff comprising admixing a feed component with a feedadditive composition according to any one of claims 1-21.
 81. A premixcomprising a feed additive composition comprising a direct fed microbialin combination with a protease, a xylanase, an amylase and a phytase,and at least one mineral and/or at least one vitamin.
 82. A premixcomprising a feed additive composition according to any one of claims1-21 in combination with at least one mineral and/or at least onevitamin.
 83. A feed additive composition according to any one of claims1-21 for preventing and/or treating coccidiosis and/or necroticenteritis in a subject.
 84. A method of preventing and/or treatingnecrotic enteritis and/or coccidiosis wherein an effective amount of afeed additive composition according to any one of claims 1-21 isadministered to a subject.
 85. A feed additive composition or feed orkit or method or use or premix as defined generally herein withreference to the Figures and the Examples.